Hydrogel-based vascular lineage cell growth media and uses thereof

ABSTRACT

A medium for growing vascular lineage cells is described. The vascular lineage cell growth medium includes an oligosaccharide-based hydrogel and a growth factor that promotes vascularization by vascular lineage cells.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application No.61/259,821 filed Nov. 10, 2009, the entire contents of which are herebyincorporated by reference.

BACKGROUND

1. Field of the Invention

The invention relates to growth media for vascular lineage cells,particularly media using oligosaccharide-based hydrogels as a substratefor vascular lineage cell growth and morphogenesis.

2. Background of the Art

Generating a functional vascular network has the potential to improvetreatment for vascular disease and successful organ transplantation. Inrecent decades, postnatal vasculogenesis has been considered to be animportant mechanism for neovascularization via circulating endothelialprogenitor cells (EPCs) derived from marrow (Asahara et al., Science,vol. 275, pp. 964-967, 1997; Hill et al., New England J. Med., vol. 348,pp. 593-600, 2003). Since their discovery, marrow derived circulatingendothelial progenitor cells (EPCs) have been considered to participatein postnatal vasculogenesis (Asahara et al., 1997; Hill et al., 2003;Urbich et al., Circ. Res., vol. 95, pp. 343-353, 2004). Putative EPCshave been proposed as a potential therapeutic tool for treating vasculardisease, either through infusion to the site of vascularization(Schatteman et al., J. Clin. Invest., vol. 106, pp. 571-578, 2000; Silvaet al., Proceedings of the National Academy of Sciences, vol. 105, no.38, pp. 14347-14352, 2008) or ex vivo expansion for engineeringvascularized tissue constructs (Shepherd et al., FASEB J., vol. 20, pp.1739-1741, 2006; Au et al., Blood, vol. 111, pp. 1301-1305, 2008;Melero-Martin et al., Circulation Research, vol. 103, no. 2, pp.194-202, 2008). It has become more evident that endothelialcolony-forming cells (ECFCs), a subtype of EPCs recently indentifiedfrom circulating adult and human umbilical cord blood, expressedcharacteristics of putative EPCs (Yoder et al., Blood, vol. 109, pp.1801-1809, 2007; Hirschi et al., Arterioscler. Thromb. Vasc. Biol., vol.28, pp. 1584-1595, 2008; Yoder, M C, Journal of Thrombosis andHaemostasis, vol. 7, SUPPL. 1, pp. 49-52, 2009). These ECFCs arecharacterized by robust proliferative potential in forming secondary andtertiary colony as well as de novo blood vessel formation in vivo(Yoder, M C, Arterioscler. Thromb. Vasc. Biol., vol. 30, no. 6, pp.1094-1103, 2010; Ingram et al., Blood, vol. 105, no. 7, pp. 2783-2786,2005).

Differentiation, mobilization, and recruitment of EPCs and endothelialcells (ECs) are regulated foremost by vascular endothelial growth factor(VEGF) (Asahara Circ. Res., vol. 85, pp. 221-228, 1999; Li et al., FASEBJ., vol. 20, pp. 1495, 1497, 2006). Administration of VEGF into the siteof ischemia has been reported to induce ECs mobilization and restoreblood flow (Kava et al., J. Cereb. Blood. Flow. Metab., vol. 25, pp.1111-1118, 2005; Sun et al., J. Clin. Invest., vol. 111, pp. 1843-1851,2003). The extracellular matrix (ECM) provides critical support for ECs;their adhesion to the ECM is required for their proliferation,migration, morphogenesis, and survival—as well as, ultimately, for thestabilization of blood vessels (Davis et al., Circ. Res., vol. 97, pp.1093-1107, 2005),—through both biochemical and mechanical functions(Deroanne et al., Cardiovasc. Res., vol. 49, pp. 647-658, 2001;Sieminski et al., Cell Biochem. Biophys., vol. 49, pp. 73-83, 2007);Mammoto et al., Nature, vol. 457, pp. 1103-1108 2009). Matrix elasticityhas been reported to induce stem cell differentiation and morphologicalchanges (McBeath et al., Dev. Cell., vol. 6, pp. 483-495, 2004; Engleret al., Cell, vol. 126, pp. 677-689, 2006). Changes in physicalinteractions between cell surface integrins and the ECM, due toalterations in ECM elasticity, regulate cell shape and cytoskeletalstructure (Ingber et al., J. Cell Biol., vol. 109, pp. 317-330, 1989;Matthews et al., J. Cell Sci., vol. 119, pp. 508-518, 2006; Chen et al.,Science vol. 276, pp. 1425-1428, 1997). Mechanical forces exerted by ECson the matrix stimulate capillary growth in vivo (Moore et al., Dev.Dyn., vol. 232, pp. 268-281, 2005) and formation of capillary-likestructures (CLSs) in vitro (Davis et al., Exp. Cell Res., vol. 216, pp.113-123, 1995; Ingber et al., Cell, vol. 58, pp. 803-805, 1989).Recently, matrix elasticity has been reported to modulate the expressionof VEGF receptor 2 (VEGFR2) (Mammoto et al., Nature, vol. 457, pp.1103-1108, 2009), and biomechanical forces alone were sufficient tomediate vascular growth in vivo independent of endothelial sprouting(Kilarski et al., Nat. Med., vol. 15, pp. 657-664, 2009).

Tube morphogenesis is essential for the development of a functionalcirculatory system [24,25]. Longitudinal vacuoles that appeared to beextruded and connected from one cell to the next were first described byFolkman and Haudenschild (Folkman et al., Nature, vol. 288, pp. 551-556,1980). These observations were confirmed and extended by later studiesshowing that intracellular vacuoles arise from events downstream ofintegrin-ECM signaling interactions, where lumen formation is mediatedthrough the activation of the Rho GTPase Cdc42 (Kamei et al., Nature,vol. 442, pp. 453-456, 2006; Bayless et al., J. Cell Sci., vol. 115, pp.1123-1136, 2002; Davis et al., Exp. Cell Res., vol. 224, pp. 39-51,1996; Iruela-Arispe et al., Dev. Cell, vol. 16, pp. 222-231, 2009).Moreover, at the site of neovascularization, activated membrane type1-matrix metalloproteinase (MT1-MMP) activates pro-MMPs at thepericellular area, which digest the ECM and allow EC migration andtubulogenesis (Collen et al., Blood, vol. 101, pp. 1810-1817, 2003;Galvez et al., J. Biol. Chem., vol. 276, pp. 37491-37500). Such matrixremodeling by MMPs is implicated in various pathological conditionsincluding atherosclerosis, inflammation, and ischemia (Romanic et al.,Stroke, vol. 29, pp. 1020-1030, 1998; Galis et al., J. Clin. Invest.,vol. 94, pp. 2493-2503, 1994).

Vascular regeneration and repair are complex processes, which requireEPCs to break down the extracellular matrix (ECM), migrate,differentiate, and undergo tube morphogenesis. In the last decadesunderstanding of the role of ECM in vascular morphogenesis has beenwidely expanded due to well defined in vitro angiogenesis models.Natural ECM such as matrigel, collagen, and fibrin gels have been widelyused to study the molecular mechanisms that regulate endothelial cells(ECs) tubulogenesis (Davis et al., Birth Defects Research Part C—EmbryoToday: Reviews, vol. 81, no. 4, pp. 270-285 2007; Kniazeva et al., Am.J. Physiol. Cell Physiol., vol. 297, no. 1, pp. C179-187, 2009), as wellas to transplant vascular progenitor cells, such as ECFCs (Critser etal., “Collagen matrix physical properties modulate endothelial colonyforming cell-derived vessels in vivo.” Microvascular Research, In Press)and EPCs and mesenchymal stem cells (Au et al., Blood, vol. 111, pp.1302-1305, 2008; Au et al., Blood, vol. 111, no. 9, pp. 4551-4558, 2008;Melero-Martin et al., Circulation Research, vol. 103, no. 2, pp.194-202, 2008) to generate vascular networks in vivo. However, theinherent chemical and physical properties of these natural materialshave limited their manipulation for vascular tissue engineering.

The ECM contains instructive physical and chemical cues required for adelicate balance between various factors and cells to guide vascularassembly (Davis, G E, Am J Physiol Heart Circ Physiol, vol. 299, pp.H245-H247, 2010; Sacharidou et al., Blood, vol. 115, no. 25, pp.5259-5269, 2010; Deroanne et al., Cardiovasc. Res., vol. 49, pp.647-658, 2001; Sieminski et al., Cell. Biochem. Biophys., vol. 49, pp.73-83, 2007; Kniazeva et al., Am. J. Physiol. Cell Physiol., vol. 297,no. 1, pp. C179-187, 2009; Mammoto et al., Nature, vol. 457, pp.1103-1108, 2009; Stratman et al., Blood, vol. 114, no. 2, pp. 237-47,2009). Depending on their spatial and temporal distribution throughoutthe body, each ECM component can have a different role in angiogenesis.HA and fibronectin, which are major components of embryonic ECM, arevital vascular regulators during embryogenesis (Toole, B P, Semin. Cell.Dev. Biol., vol. 12, no. 2, pp. 79-87, 2001; Toole, B P, Nat Rev Cancer,vol. 4, no. 7, pp. 528-539, 2004); while, collagen and laminin, whichare abundant in adult ECM, are crucial for maintaining vascularhomeostasis in adults (Davis et al., Current Opinion in Hematology, vol.15, no. 3, pp. 197-203, 2008). Natural ECM, like collagen, fibrin, andmatrigel, has been used both as model to study vascular morphogenesisand as scaffold to deliver vascular construct. However the use ofnatural ECM hydrogels has been limited due to their inherent physicaland chemical properties. Moreover, their clinical usage has beenhampered due to problems associated with complex purification processes,pathogen transfer, and immunogenicity.

SUMMARY

Synthetic biomaterials, which are xeno-free and more-clinically relevantfor regenerative medicine, have been suggested as an alternative (Lutolfet al., Proc. Natl. Acad. Sci. U.S.A., vol. 100, no. 9, pp. 5413-5418,2003; Ehrbar et al., Circ Res, vol. 94, no. 8, pp. 1124-1132, 2004).Unlike natural ECM, these synthetic biomaterials can potentially beengineered to provide an instructive microenvironment, which couldorchestrate the complex stages of vascular morphogenesis (Lutolf et al.,Nature Biotechnology, vol. 23, no. 1, pp. 47-55, 2005). Althoughprevious studies have used self-assembling peptide (Sieminski et al.,Cell. Biochem. Biophys., vol. 49, pp. 73-83, 2007; Sieminski et al.,Journal of Biomedical Materials Research, vol. 87A, no. 2, pp. 494-504,2008) and PEG hydrogels (Moon et al., Biomaterials, vol. 31, no. 14, pp.3840-3847, 2010; Leslie-Barbick et al., Journal of Biomaterials Science,Polymer Edition, vol. 20, no. 12, pp. 1763-1779, 2009) to generatevascular networks in vitro, very few studies have investigated vascularnetworks assembly within such synthetic biomaterials (Moon et al.,Biomaterials, vol. 31, no. 14, pp. 3840-3847, 2010; Sieminski et al.,Exp. Cell. Res., vol. 297, pp. 574-584, 2004). The present inventionincludes synthetic biomaterials for vascular morphogenesis that have notbeen previously available.

Hydrogels with defined compositions and tunable elasticity are used topromot in vitro tube morphogenesis. These hydrogels serve as substratesof varying stiffness, that can affect the kinetics of EPC tubulogenesis.Viscoelasticity measurements during in situ gelation demonstrate threedistinct substrate stiffness profiles: rigid, firm, and yielding thathave varying effects on tube morphogenesis. While low levels of VEGFallow EPCs to spread on all substrates, higher levels of VEGF may betterinitiate tube morphogenesis and activate MMPs, whose expression declineswith the decrease of matrix stiffness. Increased tubemorphogenesis—including CLS progression, vesicle formation, expansion(in both size and number) and fusion to lumens—is better promoted withdecreased substrate rigidity. RNA interference (RNAi) studies furthershow that MT1-MMP and Cdc42 further promote tube morphogenesis fromEPCs.

Synthetic acrylated HA (AHA) hydrogels are example substrates to controlvascular morphogenesis of ECFCs. Adhesion and cleavability promotershelp efficiently form vascular networks. This can improve kinetics andECFC material interactions during various stages of morphogenesis, whichinvolve vacuoles and lumen formation; branching and sprouting; andnetwork formation and growth. Development and use of hydrogels alsorequires consideration of the molecular mechanism that regulates ECFCsvascular assembly within the hydrogel. Following transplantation, humanvascular networks formed within AHA hydrogels according to the inventionanastomize with the host circulation and establish blood flow in thehydrogels.

Embodiments include an vascular lineage cell growth medium including ahydrogel with a stiffness that allows formation of capillary-likestructures (CLSs) with extended vacuoles and open lumens from vascularlineage cells. The hydrogel is a crosslinked mixture of anoligosaccharide and a crosslinking moiety, and is cleavable by anenzyme. The medium further includes a growth factor in an amountsufficient to promote vasculogenesis and tube formation, and sufficientto stimulate MMP production in vascular lineage cells. In someembodiments, the hydrogel, or crosslinked mixture, further includes anadditional ingredient selected from the group consisting of gelatin,collagen, fibrin, and laminin.

In some embodiments, the crosslinking moiety is cleavable by the enzyme.In some embodiments the crosslinking moiety is a peptide. And in someembodiments, the enzyme is a matrix metalloproteinase. Examples ofmatrix metalloproteinases include MMP-1, MMP-2 or MMP-10. In someembodiments, the peptide crosslinking moiety includes the amino acidsequence GCRDGPQGIWGQDRCG.

In some embodiments, the hydrogel, or crosslinked mixture, furtherincludes an adhesion promoter in an amount sufficient to promotevascular lineage cell adhesion to the hydrogel and promote vacuoleformation in vascular lineage cells. In some embodiments, the adhesionpromoter is bonded to the oligosaccharide.

In some embodiments, the stiffness of the hydrogel has a Young's modulusbetween about 10 Pa and about 500 Pa. In some embodiments, the hydrogelhas a Young's modulus less than about 250 Pa.

In some embodiments, the oligosaccharide is modified with an acrylgroup. In other embodiments, the oligosaccharide is modified with athiol group. In some embodiments, oligosaccharide is hyaluronic acid,dextran, alginate, or chitosan. In some embodiments, the oligosaccharideis hyaluronic acid. The oligosaccharide may be acrylated hyaluronicacid.

In some embodiments, the growth factor is VEGF, TNFα, SDF1-α, bFGF,angiopoetin-1, PDGF, TGF-β, PIGF or combinations thereof. In someembodiments, the growth factor is VEGF. In some embodiments, the mediumincludes more than one growth factor. For instance, the growth factorsmay be VEGF and at least one additional growth factor.

In some embodiments, the growth medium further includes vascular lineagecells. Vascular lineage cells may be, for example, endothelial cells,endothelial progenitor cells, or endothelial colony forming cells.

The growth medium may be prepared by chemically bonding an adhesionpromoter to the oligosaccharide, combining the chemically bondedadhesion promoter and oligosaccharide with at least one growth factor,adding an enzymatically cleavable crosslinking peptide moiety, andcuring the combination to form a hydrogel where the oligosaccharide ishyaluronic acid that is from about 20 to about 90% acrylate modified andis present in an amount of about 1 wt % to about 4 wt %. The adhesionpromoter is present at a concentration of from about 0.1 mM to about 20mM. The adhesion promotor may be, for example, an RGD sequencecontaining peptide. The enzymatically cleavable crosslinking peptide ispresent at a concentration between about 1 mM to about 8 mM. The growthfactor is present at a concentration of from about 25 ng/ml to about 200ng/ml.

Embodiments include methods of inducing vascularization by culturingvascular lineage cells in a medium comprising a hydrogel and a growthfactor, where the hydrogel has a Young's modulus between about 10 Pa toabout 500 Pa and the hydrogel comprises a crosslinked mixture of anoligosaccharide and a crosslinking moiety. The vascular lineage cellsmay undergo vascularization within the hydrogel or on the surface of thehydrogel.

In some embodiments, the hydrogel is enzymatically cleavable. This meansthat a chemical bond within the hydrogel may be cleaved by one or moreenzymes. In some embodiments, the hydrogel may be cleaved by a matrixmetalloproteinace (MMP). Examples of matrix metalloproteinases includeMMP-1, MMP-2, and MMP-10.

In some embodiments, the oligosaccharide is acrylated hyaluronic acid.

In some embodiments of the method, the hydrogel further includes andadhesion promoter.

In some embodiments of the method, the medium is prepared by chemicallybonding an adhesion promoter to the oligosaccharide, combining thechemically bonded adhesion promoter and oligosaccharide with at leastone growth factor, adding an enzymatically cleavable crosslinkingpeptide moiety, and curing the combination to form a hydrogel. Theoligosaccharide is hyaluronic acid that is from about 20 to about 90%acrylate modified and is present in an amount of about 1 wt % to about 4wt %. The adhesion promoter is present at a concentration of from about0.1 mM to about 20 mM and may be an RGD sequence containing peptide. Theenzymatically cleavable peptide crosslinking moiety is present at aconcentration of from about 1 mM to about 8 mM. The growth factor ispresent at a concentration of from about 25 ng/ml to about 200 ng/ml.

Embodiments include methods of preparing a vascular network bychemically bonding an adhesion promoter to an oligosaccharide andcombining the chemically bonded adhesion promoter/oligosaccharide withat least one growth factor and vascular lineage cells. A crosslinkingmoiety capable of forming a hydrogel with the oliosaccharide is added tothe combination, followed by curing the combination to form a hydrogel.Vascular lineage cells may be added before, during or after curing. Thevascular lineage cells are incubated to form a vascular network.

The oligosaccharide may be, for example, hyaluronic acid, dextran,alginate, or chitosan. In some embodiments, the oligosaccharide ishyaluronic acid or acrylated hyaluronic acid.

In some embodiments, the crosslinking moiety is cleavable by a matrixmetalloproteinase. This means that a chemical bond within thecrosslinking moiety may be cleaved by at least one matrixmetalloproteinase enzyme. Examples of matrix metalloproteinases include,for example, MMP-1, MMP-2 or MMP-10. In some embodiments, thecrosslinking moiety is a peptide that includes the sequence the sequenceGCRDGPQGIWGQDRCG.

In some embodiments, the adhesion promoter is a peptide that includes anRGD sequence and a reactive functional group that is capable of bondingthe adhesion promoter to the oligosaccharide.

The growth factor may be, for example, VEGF, TNFα, SDF1-α, bFGF,angiopoetin-1, PDGF, TGF-β, PIGF or combinations thereof

Embodiments include methods where the oligosaccharide is acrylatedhyaluronic acid, and the crosslinking agent is cleavable by a matrixmetalloproteinace (MMP). The adhesion promoter is a peptide thatincludes an RGD sequence and a reactive functional group that is capableof bonding the adhesion promoter to the acrylated hyaluronic acid. Insome embodiments of the method the oligosaccharide is hyaluronic acidthat is from about 20% to about 90% acrylate modified and is present inan amount of about 1 wt % to about 4 wt %. The adhesion promoter ispresent at a concentration of from about 0.1 mM to about 20 mM. Thecrosslinking moiety is an enzymatically cleavable peptide and is presentat a concentration of from about 1 mM to about 8 mM. The growth factoris present at a concentration of from about 25 ng/ml to about 200 ng/ml;and the epithelial cell density is from about 2 million cells/mL toabout 10 million cells/mL.

In some embodiments, the hydrogel has a Young's modulus between about 10Pa and about 500 Pa. In some embodiments, the hydrogel has a Young'smodulus less than about 250 Pa.

Embodiments include methods of growing blood vessels in vitro byincubating vascular lineage cells in a medium as described herein.

Embodiments include methods of promoting vascular growth in a subject bycontacting the subject with a medium or hydrogel containing vascularlineage cells, as described herein. The vascular lineage cells may bepartly or wholly vascularized.

DESCRIPTION OF THE DRAWINGS

FIG. 1: Viscoelasticity of hydrogels. (1A). Microrheology measurementsof HA:gelatin in a 1:1 volume ratio with 1%, 0.4%, and 0.1% (w/v) ofPEGDA crosslinker over 24 hours of gelation show three distinct profilesof hydrogel mechanics: rigid, firm and yielding. Values shown aremeans±SD for Young's modulus (E) over 24 hours during the in situgelation (see FIG. 7 for elastic modulus (G′) data). (1B) After 12hours, the values of Young's modulus (stiffness) were: 650±180 Pa forrigid hydrogel, 75±40 Pa for firm hydrogel, and 10±2 Pa for yieldinghydrogel. (1C) After an additional 12-hour gelation period, a slightincrease in stiffness was observed in all three hydrogels with valuesof: 780±240 Pa, 85±40 Pa, and 15±3 Pa, respectively.

FIG. 2: High VEGF concentrations were required for CLS formation fromCB-EPC. (2A) EPCs were seeded on rigid, firm, and yielding substratesfor 12 hours supplemented with 1 ng/ml (low) VEGF (upper panel) andformed CLSs when supplemented with 50 ng/ml (high) VEGF (lower panel),as demonstrated by fluorescence microscopy of F-actin (green) and nuclei(blue). Real Time RT-PCR revealed a significant increased expression of(2B) MT1-MMP, (2C) MMP-1, and (2D) MMP-2 in response to 50 ng/ml VEGF(high) concentration for EPCs cultured on the rigid, firm, and yielding.As the matrix substrate is reduced, EPCs cultured in media supplementedwith 50 ng/ml (high) VEGF showed a decrease in expression of (2E)MT1-MMP, (2F) MMP-1, and (2G) MMP-2 which fitted a linear trend with R2values 0.80, 0.61, and 0.94, respectively. Significance levels were setat: *p<0.05 and **p<0.01. Scale bar is 100 μm.

FIG. 3: Substrate mechanics affects CLS phenotype. Metamorph analysis ofCLSs revealed a significant increase of mean tube length (3A) and meantube area (3B) as substrate stiffness decreased. (3C) Thickened CLSsformed on yielding substrates. Confocal analysis of nuclei (blue),VE-Cad (red), and lectin (green) further revealed (3D) branching and(3E) hollowing in tubular structures formed on the yielding substrate.Significance levels were set at:*p<0.05, **p<0.01, and ***p<0.001. Scalebars are 20 μm.

FIG. 4: Substrate mechanics regulates vacuole and lumen formation. TEManalyses of CLSs formed after 6 hours showed vacuoles (V) forming on allsubstrates (S), while EPCs formed two to three layers on rigid substrate(4A), extended on firm substrate (4B), and formed an elongated singlelayer on yielding substrates (4C). After 12 hours, EPCs grew longer onall substrates and contained many vacuoles (V) on rigid (4D) substrate(S), enlarged vacuoles with occasionally noted lumens (L) on firm (4E)substrate, and open lumens (L) in complex, lengthened cellularstructures on yielding (4F) substrates. Cell nucleus is indicated in N.Scale bars are 10 μm in A and 2 μm in B.

FIG. 5: MT1-MMP and Cdc42 required for CLS formation from EPC. EPCstransfected with the indicated siRNA were seeded on rigid (5A and 5B),firm (5C and 5D), and yielding (5E and 5F) substrates and supplementedwith 50 ng/ml VEGF for 12 hours with no indication of CLS formation asdemonstrated by fluorescence microscopy of VE-cad (red) and nuclei(blue). Scale bar is 100 μm.

FIG. 6: Model for mechanics and VEGF co-regulation of tube morphogenesis(cellular and molecular level). VEGF initiates angiogenesis, with EPCassembly into a chain, by inducing MMP activation (6A), which furtherallows EPCs to elongate on substrate within 2-6 hours (6B). Substratemechanics activates Cdc42 (6A), resulting in intracellular vacuoleformation (6B), extension, and fusion to lumens after 12 hours (6B and6C).

FIG. 7: Viscoelasticity of hydrogels. Microrheology measurements ofHA:gelatin over 24 hours of gelation show three distinct profiles ofhydrogel mechanics: rigid, firm and yielding. Values shown are means±SDfor elastic modulus (G′) over 24 hours during the in situ gelation.

FIG. 8: Time interval images of EPCs on rigid, firm, and yieldingsubstrates. Rapid chain assembly and CLS formation on softer substratesalong the 12 hour culture period. Scale bar is 100 μm.

FIG. 9: RNAi for MT1-MMP and Cdc42. Real-time RT-PCR analysis of siRNAtransfected CB-EPCs shows significant suppression of MT1-MMP (9A) orCdc42 (9B) compared to controls (Luciferase-transfected EPCs).Significance levels were set at *p<0.05, and ***p<0.001, respectively.Western blot analysis shows suppression of MT1-MMP (9C) or Cdc42 (9D) atprotein level compared to Luciferase control. (9E) Light microscopeimages of CB-EPCs transfected with Luciferase, MT1-MMP, or Cdc42 seededon rigid, firm, and yielding substrates for 12 hours. Scale bar is 100μm.

FIG. 10: AHA-hydrogels: study strategy and material characterization.Schematic of study strategy (10A); NMR graph (10B) demonstrate %acrylate modification of AHA hydrogels. Polymerized AHA hydrogel (3 wt%) in a disc shape (10C) as used in this study.

FIG. 11: Determining RGD and MMP requirements for ECFC morphogenesis.RGD dose-dependent vacuolation of ECFC is demonstrated by vacuoleformation kinetics (11A) and represented LM and vacuole vital stain FM4-64 (cyan) images at 24 hours (11B). Scale bars are 100 μm. Insertdemonstrate high magnifications of single cells with abundant vacuolesin 3.7 mM RGD. Scale bars are 20 μm. Both RGD adhesion sites andMMP-degradable crosslinker for vacuolation of ECFC are demonstrated byvacuole formation kinetics (11C) and represented LM and vacuole vitalstain FM 4-64 (cyan) images at 24 hours (11D). Scale bars are 50 μm.

FIG. 12: ECFC encapsulated within AHA gels (Day0-Day1). Vacuoleformation observed few hours after cells encapsulation using: LM imaging(12A) and higher magnification of vacuolated cells (indicated byarrowheads; 12B); vacuole vital stain FM 4-64 (12C, cyan; nuclei inblue) of encapsulated cells and higher magnification of a vacuolatedcell ((indicated by arrowheads; insert); and TEM high resolutionrepresentative image (12D) of a single rounded encapsulated cell. Scalebars are 100 μm Increased number and size of vacuole and merging intolarge lumen detected by day 1 of culture, as indicated by: LM imaging(12E) and higher magnification showing merging lumen (indicated by thearrows; 12F); vacuole vital stain FM 4-64 (12G, cyan; nuclei in blue)and higher magnification focused on cell containing large vacuole(indicated by the arrowhead; insert). Scale bars are 50 μm; and TEM highresolution representative image of a encapsulated cell with apparentvacuoles (12H). N=nucleus, V=vacuoles, L=lumen, H=hydrogels. Scale baris 20 μm.

FIG. 13: ECFC within AHA gels (Day 2). Progression in tubulogenesisthrough branching and sprouting and hydrogel degradation are observedusing: LM imaging of encapsulated cells on day 2 (13A) and highermagnification focusing on branching networks (indicated by arrow; 13B)scale bars are 100 μm; vacuole vital stain FM 4-64 (cyan; nuclei inblue) on day 2 cells show representative images of branching (13C) andsprouting cells (13D). Scale bars are 50 μm; and TEM high resolutionrepresentative images of: a cell with degraded surroundings (13E); (ii)elongated cell morphology (13F and 13G); and guiding channels ofdegraded hydrogel formed between adjacent cells (13H, indicated byarrow). N=nucleus, V=vacuoles, L=lumen, H=hydrogels. Scale bar is 20 μm.

FIG. 14: Complex vascular networks formed by ECFC within AHA hydrogels(Day 3). Growth of comprehensive vascular network is demonstrated usingLM images at low magnification (14A), while i (14B) and ii (14C) arehigh magnification of white boxes (arrow indicating branched andelongated vascular networks). Scale bars are 100 μm confocal analysis ofvacuole vital stain FM 4-64 (14D, cyan; nuclei in blue) demonstratelarge lumen within the networks, which is also detected using orthogonalview (indicated by arrowhead; 14E). Scale bars are 50 μm. TEM highresolution imaging demonstrates cross sectioned of matured vascular tubenetworks with enlarged lumen (14F). Scale bar is 20 μm.

FIG. 15: Cell and material interaction. Visco elasticity measurementsrevealed decrease in hydrogels stiffness along the 3 days cultureperiod, reaching 40 Pa in hydrogel encapsulated with ECFCs (15A). SEMimages of Day 0 (15B) show rounded cells in mesh ultrastructure of theAHA hydrogels (i-iii are low to high magnification) and of Day 3 (15C)show spreading cells and formation of guiding microchannels (i-iii arelow to high magnification). Real time RT-PCR analysis show increasedexpression of MT-1 MMP, MMP1, MMP2, Hyal 2 and hyal 3 of encapsulatedECFCs along the 3 days culture period (15D). AHA degradation is detectedby cumulative percentage of released VEGF (15E) and by increased uronicacid in the media along the culture period (15F).

FIG. 16: Molecular regulation of vascular morphogenesis in AHA gels.Integrin blocking assay revealed that RGD-dependent vacuolation andlumen formation within AHA hydrogels are regulated by αVβ3 and α5β1integrins, but not by, α2β1 integrins (16A). Real time RT-PCR analysisshow downregulating expression of αV, α5, β1 and β3, along the cultureperiod of encapsulated ECFCs (16B). Confocal analysis (16C) revealed themembrane localization of MMP-1, and MMP-2 (both in green; actin in red;nuclei in blue) in the branching and sprouting ECFC (high magnificationshowed in the inserts). Scale bars are 50 μm. Real time RT-PCR analysisshow increase expression of TIMP-2 and TIMP-3 by encapsulated ECFCsalong the 3 days culture period (16D).

FIG. 17. Functionality of vascular networks. Nude mice: Histologicalanalysis of implanted vascular networks after 1 week (17A) and 2 week(17B) in vivo. The microvessels were positive for human CD31 (17C) andmouse-α-SMA (17D).

DETAILED DESCRIPTION

Some embodiments of the current invention are discussed in detail below.In describing embodiments, specific terminology is employed for the sakeof clarity. However, the invention is not intended to be limited to thespecific terminology so selected. A person skilled in the relevant artwill recognize that other equivalent components can be employed andother methods developed without departing from the broad concepts of thecurrent invention. All references cited herein are incorporated byreference as if each had been individually incorporated. Headings usedherein are provided for clarity and organizational purposes only, andare not intended to limit the scope of the invention.

Definitions

By “modifies” is meant alters. An agent that modifies a cell, substrate,or cellular environment produces a chemical or biochemical alteration ina component (e.g., polypeptide, oligonucleotide, oligosaccharide, orpolymer). Modifying a chemical or biochemical entity means that amoiety, for example, a reactive functional group, is formed or added tothe entity

By “subject” is meant an animal. In some embodiments, a subject may be amammal, including, but not limited to, a human or non-human mammal, suchas a rodent, mouse, bovine, equine, canine, ovine, or feline.

By “therapeutic delivery device” is meant any device that provides forthe release of a therapeutic agent.

As used herein, the terms “treat,” “treating,” “treatment,”“therapeutic” and the like refer to reducing or ameliorating a disorderand/or symptoms associated therewith. It will be appreciated that,although not precluded, treating a disorder or condition does notrequire that the disorder, condition or symptoms associated therewith becompletely eliminated.

Abbreviations used herein include: Hyaluronic Acid (HA); acrylatedhyaluronic acid (AHA); Endothelial Cells (EC); Endothelial ProgenitorCells (EPC); Endothelial Colony Forming Cells (ECFC); ExtracellularMatrix (ECM); Matrix Metalloproteinase (MMP); Capillary-like Structures(CLS); Vascular Endothelial Growth Factor (VEGF); Vascular EndothelialGrowth Factor Receptor (VEGFR); Stromal-cell Derived Factor (SDF); TumorNecrosis Factor (TNF).

Growth Medium

Embodiments of the invention include a growth medium for vascularlineage cells. The growth medium includes a hydrogel, which is acrosslinked mixture of an oligosaccharide and a crosslinking moiety. Thegrowth medium further includes a growth factor.

As used herein, a crosslinked mixture of oligosaccharide andcrosslinking moiety means that an oligosaccharide molecule is bonded,i.e. by a covalent bond, to two or more crosslinking moieties, and acrosslinking moiety is bonded, i.e. by a covalent bond, to two or moreoligosaccharide molecules. The crosslinked compounds form a matrix inthe form of a hydrogel.

Oligosaccharides used to form the gels of the invention will have two ormore functional groups capable of reacting with functional groups on thecrosslinking moiety to form a crosslinked matrix. The functional groupson the oligosaccharide may be the same or different, but should both beable to react with functional groups on the crosslinking moiety.

In some embodiments, the reactive functional groups on theoligosaccharide are all of the same type. In some embodiments, thereactive functional group on the oligosaccharide are acryl groups. Insome embodiments, the reactive functional group on the oligosaccharideare thiol groups.

In some embodiments, the reactive functional groups on the crosslinkingmoiety are all of the same type. In some embodiments, the reactivefunctional groups on the crosslinking moiety are acryl groups. In someembodiments, the reactive functional group on the crosslinking moietyare thiol groups.

Generally, the reactive functional group on the oligosaccharide andcrosslinking moiety will not be the same type, because they must be ableto react with each other to form the crosslinked mixture. In otherwords, if the reactive functional group on the oligosaccharide is anacryl group, the reactive functional group on the crosslinking moietymay be, for example, a thiol group. Likewise, if the reactive functionalgroup on the oligosaccharide is a thiol group, the reactive functionalgroup on the crosslinking moiety may be, for example, an acryl group.The thiol group may react with the acryl group to form a thioether, viaa Michael-type addition reaction. Examples of complementary functionalgroups that may be placed on the oligosaccharide or crosslinking moietyinclude: thiol and acrylate, thiol and maleimide, amine and aldehyde,amine and succinic anhydride, among others. Other suitable complementaryfunctional groups will be apparent to one of ordinary skill in the art.

In some embodiments, an additional ingredient is included in thecrosslinked mixture. In other words, another component that is bonded totwo or more crosslinking moieties, or two or more oligosaccharidemoieties is included in the hydrogel. The additional ingredient can be,for example, a different oligosaccharide, a peptide, an oligonucleotide,or a polymer, such as a polyacrylamide, polyester, or polyethyleneglycol. As is apparent, the additional ingredient will have a reactivefunctional group that reacts with either the oligosaccharide or thecrosslinking moiety, or both (i.e. if the additional ingredient has twodifferent functional groups). In some embodiments, the additionalingredient has a reactive functional group of a single type, forexample, thiol or acrylate. Examples of additional ingredients include,for example, gelatin, collagen, fibrin and laminin. In some embodiments,the additional ingredient is gelatin.

The medium having the hydrogel described above may include additionalcomponents. In general, the medium will be prepared in water, or buffer.Examples of suitable buffers include phosphate buffered saline (PBS) andtriethanolamine-buffered saline (TEOS). Other suitable buffers will beapparent to one of ordinary skill.

Other ingredients and additives commonly used in cell culture media mayalso be included in the medium. Ingredients typically used in cellculture media, particularly for culture media for vascular lineage cellsmay be included. For example, additional ingredients may include,heparin, endothelial cell growth supplement (ECGS), or fetal calf serum.

Growth Factor

The medium also includes a growth factor. A growth factor promotes cellgrowth or propagation, or may induce expression of genes within thecell. Examples of growth factors include: Vascular Endothelial GrowthFactor (VEGF); Stromal-cell Derived Factor (SDF), such as SDF1-α; TumorNecrosis Factor (TNF), such as TNF-α; Basic Fibroblast Growth Factor(bFGF); Epidermal Growth Factor (EGF); angiopoetin (Ang), such asangiopoetin-1 (Ang-1); Platelet Derived Growth Factor (PDGF);Transforming Growth Factor (TGF), such as TGF-β; and Placental GrowthFactor (PIGF). Other growth factors are known, and may be used in themedium. More than one growth factor may be used in combination. In someembodiments, the medium includes VEGF and at least one other growthfactor. In some embodiments, the medium includes VEGF, SDF1-α, andTNF-α.

The growth factor is present in the medium at an effectiveconcentration. In general, the effective concentration will bedetermined experimentally and will be within the level of ordinaryskill. If there is more than one growth factor, each growth factor maybe present in a different concentration, independently determined. Insome embodiments, the concentration of one or more growth factors, orthe total concentration of growth factors, may be between about 25 ng/mLand about 200 ng/mL. In other embodiments, the concentration may bebetween about 35 ng/mL and about 100 ng/mL, or between about 40 ng/mLand about 75 ng/mL. In some embodiments the growth factor is present ina concentration greater than about 25 ng/mL, greater than about 30ng/mL, greater than about 35 ng/mL, greater than about 40 ng/mL, orgreater than about 45 ng/mL. In some embodiments, the growth factor ispresent in the medium in a concentration less than about 200 ng/mL, lessthan about 150 ng/mL, less than about 100 ng/mL, or less than about 75ng/mL. In some embodiments, the growth factor is present in aconcentration of about 50 ng/mL.

Oliosaccharide

Oligosaccharides used in the hydrogels include, for example, hyaluronicacid, dextran, alginate or chitosan. Other oligosaccharides will beapparent to one of ordinary skill. In some embodiments, theoligosaccharide is hyaluronic acid.

As discussed previously, the oligosaccharide includes functional groupsthat reacts with the crosslinking moiety to form the hydrogel. In someembodiments, the oligosaccharide is modified to attach, i.e. by acovalent bond, a suitable functional group. For example, if the reactivefunctional group is an acryl group, the oligosaccharide may be treatedwith an acrylating agent, such as, for example, acryloyl chloride, toadd acryl groups to the oligosaccharide. Suitable methods for modifyingthe oligosaccharide to attach a suitable reactive functional group willbe apparent to one of ordinary skill in the art.

To form a hydrogel, the oligosaccharide must have at least two reactivegroups. When a modified oligosaccharide is used, at least two of thesaccharide units of the oligosaccharide are modified to have a reactivefunctional group. When the oligosaccharide is modified, for example, byan acrylate group, the extent of modification may be varied by varyingthe ratio between the oligosaccharide and the modifying reagent. In someembodiments, more than about 20% of the saccharide units in theoligosaccharide are modified. In other embodiments, more than about 30%,more than about 40% or more than about 50% of the saccharide units inthe oligosaccharide are modified. In some embodiments, less than about90% of the saccharide units in the oligosaccharide are modified. Inother embodiments, less than about 80%, or less than bout 70% of thesaccharide units in the oligosaccharide are modified. The extent ofmodification may be determined, for example, by NMR.

In some embodiments, the oligosaccharide is hyaluronic acid (HA).Hyaluronic acid (HA), or hyaluronan, can regulate the angiogenic processby stimulating cytokine secretion and ECs proliferation (Toole, B P,Semin. Cell. Dev. Biol., vol. 12, no. 2, pp. 79-87, 2001; Toole, BP, NatRev Cancer, vol. 4, no. 7, pp. 528-539, 2004; Genasetti et al., Connect.Tissue Res., vol. 49, no. 3, pp. 120-123 2008). HA-hydrogels consistingof well-defined synthetic polymer networks not only have a high watercontent to promote cell viability, but also biophysical and biochemicalproperties similar to many soft tissues (Burdick et al.,Biomacromolecules, vol. 6, no. 1, pp. 386-391, 2005; Vanderhooft et al.,Macromolecular Bioscience, vol. 9, no. 1, pp. 20-28, 2009). HA hydrogelshave been utilized to induce vasculogenesis from differentiating humanembryonic stem cells (Gerecht et al., Proc. Natl. Acad. Sci. U.S.A.,vol. 104, pp. 11298-11303, 2007), and to determine mechanical andultrastructure properties that support in vitro vascular morphogenesis(Hanjaya-Putra et al., Journal of Cellular and Molecular Medicine, vol.14, no. 10, pp. 2436-2447, 2010). Moreover, HA hydrogels arebiocompatible, can be biodegraded by hyaluronidase, and can be designedwith a range of mechanical properties (Burdick et al.,Biomacromolecules, vol. 6, no. 1, pp. 386-391, 2005). Overall, due toits biological relevance, chemical modifiability, and ability to supportviable cells, HA hydrogels are is an excellent materials in biomimeticenvironments to control vascular morphogenesis.

In some embodiments, the oligosaccharide is hyaluronic acid that hasbeen modified with an acryl group to form acrylated hyaluronic acid(AHA). In some embodiments, about 20% to about 90% of the saccharideunits in the hyaluronic acid are modified with acryl groups. In someembodiments, about 30% to about 80% of the saccharide units in thehyaluronic acid molecule are modified with acryl groups. In someembodiments, about 50% to about 70% of the saccharide units in thehyaluronic acid molecule are modified with acryl groups.

Crosslinking Moiety

The crosslinking moiety reacts with the oligosaccharide to form thehydrogel. As discussed previously, the crosslinking moiety has at leasttwo functional groups that react with functional groups on theoligosaccharide. In some cases, the crosslinking moiety has only twofunctional groups.

The crosslinking moiety may be a different oligosaccharide, a peptide,an oligonucleotide, or a polymer, such as a polyacrylamide, polyester,or polyethylene glycol, so long as the polymer has at least two reactivefunctional groups.

In some embodiments, the crosslinking moiety is a polyethylene glycol.Polyethylene glycol can be modified to incorporate two reactivefunctional groups, one at either end of the polymer. Examples ofreactive groups include acryl groups. In some embodiments, thecrosslinking agent is polyethylene glycol diacrylate (PEGDA). Acrosslinker such as PEGDA is not biodegradable. However, oligosaccharidehydrogels used in this invention may still be broken down to promotevascularization because the oligosaccharide portion may be cleaved. Forexample, hyaluronidase can cleave saccharide bonds in hyaluronic acid,allowing a hyaluronic acid-based hydrogel to be biodegraded or topromote vascular growth.

In some embodiments, the crosslinking moiety is a peptide. In someembodiments, one or more amide bonds in the peptide may be cleavable byone or more enzymes. In some embodiments, the crosslinking moiety iscleavable by one or more matrix metalloproteinases (MMPs). Examples ofmatrix metalloproteinases include MMP-1, MMP-2 and MMP-10. In someembodiments, the crosslinking moiety comprises the peptide sequenceGCRDGPQGIWGQDRCG, which is cleavable by both MMP-1 and MMP-2. Otherexamples of peptide sequences that can be cleaved by MMPs include, forexample IKVAV, a peptide sequence derived from laminin. In facilitatingvascularization, the use of enzyme cleavable crosslinking moieties hasthe advantage of mimicing the ECM. That is, as vascularizationprogresses, the hydrogel, like the ECM, is brokendown, for example bycellular enzymes, to allow tube morphogenesis, EPC migration, and otherphysical and chemical effects.

In general, a higher amount of crosslinking moiety relative to theamount of oligosaccharide produces a stiffer hydrogel. The stiffness ofthe hydrogel may be modified by adjusting the relative amounts ofoligosaccharide and crosslinking moiety used to prepare the hydrogel.

Adhesion Promoter

In some embodiments, the crosslinked mixture further includes anadhesion promoter. The adhesion promoter is a molecule that increasescell adhesion to the hydrogel matrix. The adhesion promoter is usuallymodified to have one functional group capable of reacting with theoligosaccharide or crosslinking agent. Where the crosslinking agent hasonly two reactive groups, the adhesion promoter should have a functionalgroup that reacts with the oligosaccharide. For example, if theoligosaccharide has thiol reactive groups, the adhesion promoter mayhave, for example, an acryl functional group. If the oligosaccharide hasacryl function groups, the adhesion promoter may have, for example athiol reactive group.

In general, higher amounts of adhesion promoter relative to theoligosaccharide will produce a hydrogel with more adhesion molecules onthe surface. However, more adhesion molecules on the surface may notnecessarily increase the extent of vascular lineage cell adhesion to thehydrogel. The optimal amount of adhesion molecules may be determinedexperimentally.

In some embodiments, the adhesion promoter is a peptide. Severalpeptides that bind to cell-surface molecules on epithelial cells areknown, and any suitable peptide may be used. In some embodiments theadhesion promoter comprises the tripeptide arginine-glycine-aspartate(RGD), which binds integrin molecules on vascular lineage cells.

In natural ECM, like fibrin gel, RGD containing peptides have been shownto regulate vacuoles and lumen formation, a first step in vascularmorphogenesis (Bayless et al., Am. J. Pathol., vol. 156, no. 5, pp.1673-1683, 2000). Moreover, it has been shown that the quantity andpresentation of RGD regulates the formation of capillary networks invitro (Ingber et al., J. Cell. Biol., vol. 109, pp. 317-330, 1989).Therefore, in designing bio-mimetic AHA gels, adhesion sites within AHAhydrogels promote vacuole formation which subsequently coalescence intoan open lumen in ECFCs. This response is dose dependent.

The adhesion promoter molecule may be modified, if necessary, toincorporate a reactive functional group, such as, for example, a thiolor acryl group, to enable it to react with reactive functional groups onthe oligosaccharide or modified oligosaccharide. Any modification may beused, so long as the modification does not interfere with the ability ofthe adhesion promoter to adhere to vascular lineage cells. In someembodiments, the adhesion promoter is modified to incorporate a thiol.For a peptide adhesion promoter, such as RGD, an additional amino acidhaving a thiol reactive group, such as cysteine, may be added to thepeptide to allow it to react with acryl groups on the oligosaccharide.As used herein, RGD means peptides containing the RGD sequence, and mayinclude additional amino acids.

When RGD is used as an adhesion promoter, additional amino acids may beincluded, so long as one amino acid includes a free reactive group, suchas a thiol group. An amino acid containing a thiol group, such ascysteine, may provide the free reactive group. For example, the RGDsequence may be incorporated into a longer sequence, such as, forexample, GCGYGRGDSPG, having the RGD adhesion promoter, and a cysteine(C) with a free thiol group. Other sequences may be used, so long as thesequence includes the RGD adhesion promoter and an amino acid having afree thiol, such as a cysteine (C).

In some embodiments, the hydrogel used in the medium includes anadhesion promoter. In some embodiments, the adhesion promoter is an RGDpeptide. In some embodiments, the hydrogel is prepared with an RGDpeptide in a concentration between about 0.1 mM and about 20 mM. In someembodiments, the hydrogel is prepared with an RGD peptide in aconcentration between about 2 mM and about 10 mM, or between about 2 mMand 5 nM.

Vascular Lineage Cells

In some embodiments, the medium further includes vascular lineage cells.As used herein, vascular lineage cells include cells which becomeendothelial cells. For example, vascular lineage cells include:endothelial cells (EC), pericytes, smooth muscle cells and fibroblasts,that can be derivatives of: endothelial progenitor cells (EPC),endothelial colony forming cells (ECFC), pluripotent stem cells, othervascular progenitor cells such as those from peripheral and cord bloodprogenitors, bone marrow precursor cells, and mesenchymal derivatives,such as mesenchymal stem cells. In some embodiments, the vascularlineage cells are include endothelial cells (EC), endothelial progenitorcells (EPC), endothelial colony forming cells (ECFC), and vascular cellsfrom pluripotent derivatives, vascular progenitor cells, such as thosefrom peripheral and cord blood progenitors, bone marrow derived vascularprecursor cells, such as bone marrow endothelial progenitors; andmesenchymal derivatives, such as mesenchymal vascular cells. In someembodiments, the vascular lineage cells are endothelial cells (EC),endothelial progenitor cells (EPC), or endothelial colony forming cells(ECFC).

Endothelial progenitor cells (EPCs) in the circulatory system have beensuggested to maintain vascular homeostasis and contribute to adultvascular regeneration and repair. These processes require that EPCsbreak down the extracellular matrix (ECM), migrate, differentiate, andundergo tube morphogenesis. The ECM plays a critical role by providingbiochemical and biophysical cues that regulate cellular behavior. Usinga chemically and mechanically tunable hydrogel for tube morphogenesis invitro, allows vascular endothelial growth factor (VEGF) and substratemechanics to effectively co-regulate tubulogenesis of EPCs. In somecases, high levels of VEGF may better initiate tube morphogenesis andactivate matrix metalloproteinases (MMPs), which enable EPC migration.Under these conditions, the elasticity of the substrate affects theprogression of tube morphogenesis. With decreases in substratestiffness, decreased MMP expression is observed while increased cellularelongation, with intracellular vacuole extension and coalescence to openlumen compartments, takes place. RNAi studies demonstrate that membranetype 1—MMP (MT1-MMP) enables the movement of EPCs on the matrix and thatEPCs sense matrix stiffness through signaling cascades leading to theactivation of the RhoGTPase Cdc42. Collectively, coupled responses forVEGF stimulation and modulation of substrate stiffness regulate tubemorphogenesis of EPCs.

Endothelial colony forming cells (ECFCs) have robust proliferativepotential and can form vascular networks in vivo. ECFCs can be recruitedfrom a bone marrow niche to the site of vascularization, where cues fromthe extracellular matrix (ECM) instigate vascular morphogenesis.Although this process has been elucidated using natural matrix, thepresent invention provides for vascular morphogenesis by ECFCs in asynthetic matrix, a xeno-free scaffold which provides a morecontrollable and clinically relevant alternative for regenerativemedicine. For example, chemically and mechanically tunable hyaluronicacid (HA) hydrogels serve as three-dimensional (3D) scaffolds forvascular morphogenesis by ECFCs. Several factors considered inconjunction are used to produce a suitable hydrogel medium. First, anadhesion molecule (also referred to herein as an adhesion promoter), forexample, RGD, may be included at a concentration to induce efficientvacuoles formation and lumen formation. Integrin blocking studiesconfirm that vacuole and lumen formation are dose-dependent on adhesionmolecule concentration and recognized by ECFCs through integrin α5β1 andαVβ3. Further, integration of an enzyme cleavable crosslinker, forexample, an MMP degradable-peptide as crosslinker enables ECFCs tosprout, branch, and form complex vascular networks. Increased expressionof Hyal-1, Hyal-2, MMP-1,-2, and MT1-MMP are followed by the decrease inmatrix stiffness, suggesting that ECFCs are able to remodel the gelsthroughout the process of vascular morphogenesis. Further confocal andTEM analysis throughout the vascular morphogenesis process indicatecomplex vessel networks form with an open lumen compartment. In vivostudies in nude mice indicate that these engineered-vessels are robustand functional. Most of the microvessels at the center of the hydrogelsare positive for human CD31 and perfused with blood cells, indicativethat the implanted human vascular networks were able to anastomose withthe host vasculatures to form functional vessels (vasculogenesis). Someof the microvessels contain both human CD31+ cells and host cells,confirming that the implanted ECFCs participate in the angiogenesis ofthe host vasculatures as well.

Hydrogel Stiffness and Vasculcmenesis

The Young's modulus of the hydrogel is one indication of the stiffness.The stiffness of the hydrogel may affect the growth and/or morphology ofthe vascular lineage cells, as discussed in greater detail below. Ingeneral, the stiffness of the hydrogel can be varied by varying therelative amounts of oligosaccharide and crosslinking agent used to formthe hydrogel. In general, a higher relative amount of crosslinker willproduce a stiffer hydrogel.

In some embodiments, the Young's modulus of the hydrogel is about 500 Paor less. In some embodiments, the Young's modulus of the hydrogel isabout 300 Pa or less. In some embodiments, the Young's modulus of thehydrogel is about 250 Pa or less. In some embodiments, the Young'smodulus of the hydrogel is less than about 150 Pa, less than about 100Pa, or less than about 80 Pa. In some embodiments, the Young's modulusis greater than about 10 Pa. In some embodiments, the Young's modulus isgreater than about 20 Pa, greater than about 30 Pa, greater than about40 Pa, or greater than about 50 Pa.

Postnatal vasculogenesis has been considered to be an importantmechanism for angiogenesis via marrow-derived circulating EPCs (Asaharaet al., Science, vol. 275, pp. 964-967, 1997). In response to VEGF, EPCsare mobilized to the site of vascularization, which initiates theirproliferation and differentiation (Aicher et al., Hypertension, vol. 45,pp. 321-325, 2005). After activation, EPCs undergo a complex processthat involves migration, digestion of basement membrane, sprouting, tubemorphogenesis, and ultimately, blood vessel stabilization (Davis et al.,Circ. Res., vol. 97, pp. 1093-1107, 2005; Urbich et al., Circ. Res.,vol. 95, pp. 343-353, 2004). The ECM components play a role in thesecascades of events, which are also regulated by cytokines, integrins,and proteases. Therefore, changes in ECM mechanics may modulateproperties of EPCs. Media according to the present invention mimic thefunctionality of the various components of the ECM, thus providing forthe cascade of events needed for vascularization.

Changes in ECM mechanics lead to changes in growth factor availability,guide developmental and adaptive changes, and affect cellular fate andthe lineage commitment of stem cells (McBeath et al., Dev. Cell., vol.6, pp. 483-495, 2004; Engler et al., Cell, vol. 126, pp. 677-689, 2006).Biomechanical tension between ECs and the ECM regulates capillarydevelopment (Davis et al., Circ Res., vol. 97, pp. 1093-1107, 2005;Ingber et al., J. Cell. Biol., vol. 109, pp. 317-330, 1989; Davis etal., Exp. Cell Res., vol. 216, pp. 113-123, 1995), in which mechanicalforces exerted by the cells onto the surrounding ECM create pathways formigration that drive migratory ability and subsequent CLS formation (Loet al., Biophys. J., vol. 79, pp. 144-152, 2000; Discher et al.,Science, vol. 310, pp. 1139-1143, 2005), as well as in vivovascularization (Kilarski et al., Nat. Med., vol. 15, pp. 657-664,2009). Media according to the present invention may be adjusted to mimicECM mechanics to promote migration, CLS formation, and vascularization.

Hydrogels according to the invention are structurally and mechanicallysimilar to the native ECM of many tissues, and provide a superior matrixfor promoting cellular responses to mechanical stresses (Engler et al.,Cell, vol. 126, pp. 677-689, 2006; Discher et al., Science, vol. 310,pp. 1139-1143, 2005; Ghosh et al., Biomaterials, vol. 28, pp. 671-679,2007), as well as endothelial tubulogenesis (Sieminski et al., Cell.Biochem. Biophys., vol. 49, pp. 73-83, 2007; Sieminski et al., Exp.Cell. Res., vol. 297, pp. 574-584, 2004). Hydrogel stiffness modulatesthe ability of ECs to elongate and contract their surroundings; areduced tension between the ECs and ECM can trigger an intercellularsignaling cascade leading to cellular movement and tubulogenesis(Deroanne et al., Cardiovasc. Res., vol. 49, pp. 647-658, 2001;Sieminski et al., Cell. Biochem. Biophys., vol. 49, pp. 73-83, 2007;Sieminski et al., Exp. Cell. Res., vol. 297, pp. 574-584, 2004; Ingber DE, Circ. Res., vol. 91, pp. 877-887, 2002). Mechanical cues from the ECMand signals from growth factor receptors regulate the balance ofactivity between TFII-I and GATA2, which govern the expression ofVEGFR2, which, in turn, instigates angiogenesis (Mammoto et al., Nature,vol. 457, 1103-1108, 2009). However, elasticity range and optimapreviously reported in studies varies depending on the type of hydrogel(e.g., matrigel, collagen, polyacrylamide, or self-assembly peptide),culture system (two- vs. three-dimensional), and assay (in vivo vs. invitro) used in the study. Hydrogels used in the present invention may bereproducibly adjusted to mimic the native ECM to promote cellularresponses to mechanical stresses.

Although in vivo and in vitro studies have shown that lumenal structuresand tube formation involve the formation of intracellular vacuoles(Kamei et al., Nature, vol. 442, pp. 453-456, 2006; Bayless et al., J.Cell. Sci., vol. 115, pp. 1123-1136, 2002), the molecular mechanisms ofthese processes have been delineated only recently. Using collagen as a3D in vitro model of angiogenesis, Davis and colleagues showed that EClumenization via formation and coalescence of pinocytic intracellularvacuole involves coordinated signaling pathways: localized at cellmembranes, MT1-MMP degrades the ECM and creates a physical space tofacilitate lumen formation (Chun et al., J. Cell Biol., vol. 167, pp.757-767, 2004; Saunders et al., J. Cell Biol., vol. 175, pp. 179-191,2006), while integrin-ECM interactions initiate a cascade of downstreamsignaling to activate the Rho GTPases Cdc42 and Racl, which drive theformation of intracellular vacuoles (Bayless et al. J. Cell Sci., vol.115, pp. 1123-1136, 2002).

Molecular mechanisms are co-regulated by growth factors and ECMelasticity. MT1-MMP, which is present as an active enzyme on the cellsurface, acts directly against different ECM proteins and can activatepro-MMPs at the cell surface, which localizes MMP activity to thepericellular area (Van Hinsbergh et al., Arterioscler. Thromb. Vasc.Biol., vol. 26, pp. 716-728, 2006; Haas T L, Can. J. Physiol.Pharmacol., vol. 83, pp. 1-7, 2005). VEGF or other growth factors candirectly regulate the production of MT1-MMP, MMP-1 and MMP-2 in EPCs andactivity of MT1-MMP is required to initiate tube morphogenesis. As shownin the present invention, suppression of MT1-MMP mitigates CLS formationon firm and yielding substrates while maintaining spreading on rigidsubstrates. Thus, MT1-MMP suppressed EPCs are no longer able to degradethe surrounding ECM in order to form CLSs. On softer gels, where EPCsproduced the least amount of MMPs and have dynamic adhesions (Pelham etal., Proc. Natl. Acad. Sci. U.S.A., vol. 94, pp. 13661-13665, 1997),MT1-MMP suppression prevents them from spreading and causes them toexhibit a rounded morphology; on the rigid substrate, it allows EPCs tospread with a morphology similar to that exhibited when cultured inmedia with low levels of VEGF. The inventive medium highlights theimportance of these molecular mechanisms.

Downstream of integrin signaling, Cdc42 has been shown, using both invitro and in vivo models, to mediate vascular morphogenesis events(Kamei et al., Nature, vol. 442, pp. 453-456, 2006; Bayless et al., J.Cell. Sci., vol. 115, pp. 1123-1136, 2002). As shown in studies relatingto the present invention, inhibition of Cdc42 prevented CLS formation onall substrates, regardless of elasticity. However, while the inhibitionof Cdc42 does not prevent EPCs spreading on the rigid and firmsubstrates, they appear round on the yielding substrate. Suppression ofCdc42 prevents EPCs from physically resisting cell traction forces thatare needed on the soft gel (Discher et al., Science, vol. 310, pp.1139-1143, 2005; Ingber D E, Circ. Res., vol. 91, pp. 877-887, 2002),which result in rounded cell morphology on the yielding substrate.Furthermore, it was recently suggested that activated Cdc42 augmentedMMP-2 and MT1-MMP activity (Ispanovic et al., Am. J. Physiol. CellPhysiol., vol. 295, pp. C600-C610, 2008), which supports that Cdc42suppressed EPCs are unable to form CLSs on all substrates. The inventivemedium further establishes the importance of Cdc42 signalling.

Acrylated Hyaluronic Acid Hydrogels

In some embodiments, the hydrogel is a crosslinked mixture of acrylatedhyaluronic acid (HA) as the oligosaccharide, a thiol-terminated RGDintegrin binding peptide as the adhesion promoter, and a peptidecrosslinking moiety that is enzymatically degradable by both MMP-1 andMMP-2 that includes the sequence GCRDGPQGJ↓IWGQDRCG, where ↓ denotes thesite of cleavage.

In some embodiments, more than about 20% of the saccharide units in thehyaluronic acid are acrylated. In other embodiments, more than about30%, more than about 40% or more than about 50% of the saccharide unitsin the hyaluronic acid are acrylated. In some embodiments, less thanabout 90% of the saccharide units in the hyaluronic acid are acrylated.In other embodiments, less than about 80%, or less than bout 70% of thesaccharide units in hyaluronic acid are acrylated. The extent ofmodification may be deteremined, for example, by NMR.

In some embodiments, the AHA may be about 50% modified (50% of therepeating HA macromer contains acrylate group), and are used to form 3wt % hydrogels (FIG. 10B and 10C). Higher acrylate modification and wt %correspond to relatively dense networks and can also be used but do notas effectively support vascular morphogenesis.

Acrylated HA (AHA) hydrogels when used in media according to the presentinvention provide the dynamic cues required for network assembly ofencapsulated ECFCs (FIG. 1A). These hydrogels are formed usingsequential crosslinking from acrylated HA by reacting with athiol-terminated adhesion promoter, such as the RGD integrin-bindingpeptide (thiol group on RGD react with acrylates along the HA backbone)followed by an addition reaction with a crosslinking moiety, forexample, a thiol-terminated peptide crosslinker (thiol groups on eachend of peptide react with acrylates along the HA backbone) (Khetan etal., Soft Matter, vol. 5, no. 8, pp. 1601-1606, 2009). Example AHAhydrogels include an enzymatically degradable peptide crosslinker thatcan be cleaved by MMP-1, MMP-2, MMP-10 or others both (Lutolf et al.,Proc. Natl. Acad. Sci. U.S.A., vol. 100, no. 9, pp. 5413-5418, 2003;Seliktar et al., J. Biomed. Mater. Res. A, vol. 68, no. 4, pp. 704-716,2004), for example a peptide with the sequence GCRDGPQGJ↓IWGQDRCG, where↓ denotes the cleavage site. An adhesion promoter, such as RGD, which isthe sequence within the fibronectin molecule that mediates cellattachment, can also be included.

Methods

Embodiments include methods of inducing vascularization by culturingvascular lineage cells in a medium comprising an oligosaccharide-basedhydrogel and a growth factor, where the hydrogel has a Young's modulusbetween about 10 Pa and about 500 Pa. Any hydrogel discussed herein maybe used in the culture medium. Oligosaccharides, adhesion promoters, andcrosslinking moieties discussed herein may make up the hydrogel, andgrowth factors discussed herein may be used in the medium.

In some embodiments, the vascular lineage cells undergo vascularizationwithin the hydrogel or on the surface of the hydrogel.

In some embodiments, the hydrogel is enzymatically cleavable. In otherwords, chemical bonds within the hydrogel may be cleaved by one or moreenzymes. In some embodiments, the hydrogel is cleavable by a matrixmetalloproteinase, such as, for example MMP-1, MMP-2, or MMP-10. Acleavable crosslinking moiety, as discussed herein, will produce ahydrogel that is enzymatically cleavable. In some instances, theoligosaccharide portion may be enzymatically cleavable.

In some embodiments, the Young's modulus of the hydrogel is about 500 Paor less. In some embodiments, the Young's modulus of the hydrogel isabout 300 Pa or less. In some embodiments, the Young's modulus of thehydrogel is about 250 Pa or less. In some embodiments, the Young'smodulus of the hydrogel is less than about 150 Pa, less than about 100Pa, or less than about 80 Pa. In some embodiments, the Young's modulusis greater than about 10 Pa. In some embodiments, the Young's modulus isgreater than about 20 Pa, greater than about 30 Pa, greater than about40 Pa, or greater than about 50 Pa.

Embodiments include methods of preparing a vascular network bychemically bonding an adhesion promoter to an oligosaccharide, thencombining the chemically bonded adhesion promoter/oligosaccharide withat least one growth factor and epithelial cells. Then an enzymaticallycleavable peptide crosslinking moiety is added, and the combination iscured to form a hydrogel. The hydrogel and cells are then incubated toform a vascular network. Oligosaccharides, adhesion promoters,enzymatically cleavable peptide crosslinking moieties, and growthfactors discussed herein may be used in the method.

Embodiments include methods of growing blood vessels in vitro byincubating vascular lineage cells with a growth medium described herein.

Embodiments include methods of promoting vascular growth in a subject bycontacting the subject with a hydrogel described herein having vascularlineage cells. In this case, the vascular lineage cells may already havebegun vascularization by incubating the medium prior to contacting thesubject with the hydrogel.

Uses

Hydrogels and methods according to the invention can be used in tissueengineering scaffolds, wound care and for other purposes. Exampleembodiments of the invention include methods for promotingvascularization in vitro, creating vascular networks in engineeredtissues, use as a graft for ischemia, or as a cardiac patch/graft. Otheruses include methods of promoting burn and wound healing. Otherembodiments include methods of improving grafting, promoting injuredtissue regeneration and improving injured tissue functionality.

The generation of functional vascular networks has the potential toimprove treatment for vascular diseases and to facilitate successfulorgan transplantation. Other embodiments of the invention includemethods of increasing vascular regeneration comprising administering acomposition described above. Methods of increasing vascular regenerationcan promote, for example, burn and wound healing. As engineered tissuesbecome available, compositions and methods of the invention can be usedto promote vascularization. As shown below, use of the presentcompositions not only promote vascularization in vitro, but also allowthe new vasculature to link with existing vasculature. This can be usedto further promote tissue acceptance in the host or improve tissuegrafting.

Compositions and hydrogels including vascular lineage cells of theinvention may be used, for example, for the treatment of wounds or burnsby applying the composition to the surface of the body. The hydrogel maybe added to the body after some vascularization of the vascular lineagecells has taken place. The composition may also be administeredsubcutaneously (i.e. below the skin) to increase vascular growth orregeneration. In other cases, the composition may be implanted at aspecific location in the body, inducing vascular growth for thetreatment of, for example, ischemias.

The hydrogels described herein may be administered by any availableroute for administering hydrogels to a subject. The compositions may beformulated with at least one pharmaceutically acceptable carrierdepending on the method of administration. The compositions may beadministered, for example, parenterally, subcutaneously or topically,depending on the material to be delivered to the subject and thetargeted tissue.

In some embodiments, the composition is crosslinked prior toadministration. The crosslinked composition may be formed in aparticular shape, for example as ovoid, sphere, disc, sheet or otherstructure. Crosslinked compositions may be administered internally orexternally.

Preparation

The oligosaccharides or modified oligosaccharides described above may beprepared according to methods known in the art. Hydrogels may beprepared by combining oligosaccharides having two or more reactivefunctional groups with a crosslinking agent having two or more reactivefunctional groups. The ingredients are allowed to react until acrosslinked gel is formed.

If desired, an additional ingredient, having at least two reactivefunctional groups may be included in the reaction mixture, for example,before gel formation to modify the properties of the hydrogel. Theproportions of the ingredients may be varied to further modify theproperties of the hydrogel.

If desired, an adhesion promoter having a reactive functional group maybe reacted with the oligosaccharide prior to reaction with thecrosslinking agent, or may be included in the reaction with thecrosslinking moiety to form a hydrogel having an adhesion promoterincorporated into the crosslinked matrix.

The relative concentration of each component: oligosaccharide;crosslinker; optional additional ingredient; or adhesion promoter may bevaried to produce hydrogels with different properties such as thestiffness of the hydrogel. In some embodiments, the hydrogel is formedfrom an oligosaccharide at a concentration of about 1 wt % to about 4 wt%. In some embodiments, the hydrogel is formed from an oligosaccharideat a concentration between about 2 wt % and about 3 wt %.

In general, the number of reactive functional groups on theoligosaccharide or modified oligosaccharide will affect the amounts ofthe crosslinker, optional additional ingredient, or optional adhesionpromoter. For instance, if the oligosaccharide has more reactivefunctional groups, or the oligosaccharide is modified to a greaterextent, then higher concentrations of other components may be used. Ingeneral, the amount of crosslinker, and optional adhesion promotershould not exceed the amount of reactive functional groups on theoligosaccharide.

In some embodiments, the hydrogel is formed with a crosslinkerconcentration between about 1 mM and about 8 mM. In some embodiments,the hydrogel is formed with a crosslinker concentration between about 1mM and about 7 mM. In some embodiments, the hydrogel is formed with acrosslinker concentration between about 2 mM and about 5 mM.

In some embodiments, the hydrogel is formed with an adhesion promoterconcentration between about 0.1 mM and about 20 mM. In some embodiments,the hydrogel is formed with an adhesion promoter in a concentrationbetween about 1 mM and about 15 mM. In some embodiments, the hydrogel ifformed with an adhesion promoter in a concentration between about 2 mMand about 10 mM.

In general, higher amounts of crosslinker relative to theoligosaccharide will produce a more stiff hydrogel. In general, higheramounts of adhesion promoter relative to the oligosaccharide willproduce a hydrogel with more adhesion molecules on the surface.

Once formed, the hydrogel is combined with a growth factor to preparethe medium. Other ingredients may be included, as needed, to form aneffective vascular lineage cell growth medium. Alternatively, the growthfactor can be added prior to the formation of the hydrogel, i.e. beforeaddition of the crosslinkier. In this way, growth factors may beencapsulated within the hydrogel.

Once prepared, the medium is used to grow vascular lineage cells,including endothelial progenitor cells, or endothelial colony formingcells. Cells may be grown at a density greater than about 2 millioncells/mL, greater than about 3 million cells/mL, greater than about 4million cells/mL, or greater than about 5 million cells/mL. Cells may begrown at a density less than about 10 million cells/mL, less than about9 million cells/mL, or less than about 8 million cells/mL.

In some embodiments, the adhesion promoter is chemically bonded to theoligosaccharide prior to hydrogel formation. Reactive groups on theadhesion promoter react with reactive functional groups on theoligosaccharide to produce a chemically bonded adhesionpromoter/oligosaccharide. The adhesion promoter and oligosaccharide maybe combined in quantities such that the adhesion promotor reacts with atleast about 1%, at least about 2%, at least about 3%, at least about 5%,at least about 7%, or at least about 10% of the reactive functionalgroups on the oligosaccharide. The adhesion promoter and oligosaccharidemay be combined in quantities such that the adhesion promoter reactswith less than about 30%, less than about 25%, less than about 20% orless than about 15% of the reactive functional groups on theoligosaccharide.

Once formed, the chemically bonded adhesion promoter/oligosaccharide maybe combined with growth factors, followed by adding the crosslinkingmoiety. After the crosslinking moiety is added, the mixture forms thehydrogel.

In some embodiments, vascular lineage cells are included with themixture of chemically bonded adhesion promoter/oligosaccharide andgrowth factors, before the crosslinking moiety is added. The vascularlineage cells may be added before or after the growth factors are added.In this way a culture medium where the vascular lineage cells arepresent within the hydrogel may be prepared. In this medium, thevascular lineage cells undergo vascularization within the hydrogel, notjust on the surface.

From the foregoing description, it will be apparent that variations andmodifications may be made to the invention described herein to adopt itto various usages and conditions. Such embodiments are also within thescope of the following claims.

The recitation of a listing of elements in any definition of a variableherein includes definitions of that variable as any single element orcombination (or subcombination) of listed elements. The recitation of anembodiment herein includes that embodiment as any single embodiment orin combination with any other embodiments or portions thereof

Terms listed in single tense also include multiple unless the contextindicates otherwise.

As described herein, all embodiments or subcombinations may be used incombination with all other embodiments or subcombinations, unlessmutually exclusive.

The examples disclosed below are provided to illustrate the inventionbut not to limit its scope. Other variants of the invention will bereadily apparent to one of ordinary skill in the art and are encompassedby the appended claims. All publications, databases, and patents citedherein are hereby incorporated by reference for all purposes.

Methods for preparing, characterizing and using the compounds of thisinvention are illustrated in the following Examples. Starting materialsare made according to procedures known in the art or as illustratedherein. The following examples are provided so that the invention mightbe more fully understood. These examples are illustrative only andshould not be construed as limiting the invention in any way.

EXAMPLES

Hyaluronic acid (HA)-gelatin hydrogels were used to assess how mediumstiffness affects EPC tube morphogenesis. One advantage of HA-gelatinhydrogels is that the chemistry of the network can be controlled viareaction conditions and kept uniform between the various batches, whichis difficult or impossible to achieve with naturally derived matricessuch as matrigel and collagen. Furthermore, while enzymatic crosslinkingof natural gels such as matrigel and collagen allows studies ofincreased stiffness, chemically modified HA-gelatin hydrogels enablescontrol over crosslink density, and the ability to assess cellularresponses over a wide range of tunable mechanical stimuli. Previousstudies demonstrated that HA-gelatin hydrogels support in vivoangiogenesis (Shu et al., J. Biomed. Mater. Res. A, vol. 79, pp.902-912, 2006) and embryonic vasculogenesis (Gerecht et al., Proc. Natl.Acad. Sci. U.S.A., vol. 104, pp. 11298-11303, 2007). Thiol-modifiedHA-gelatin hydrogels enables EPC attachment. These hydrogels can bemechanically tuned using the PEGDA crosslinker while preserving uniformpresentation of cell adhesion molecules (Vanderhooft et al., Macromol.Biosci., vol. 9, pp. 20-28, 2009).

Materials and Methods Human EPCs.

Human umbilical cord EPCs isolated from outgrowth clones, provided byDr. Yoder, Indiana University School of Medicine, were expanded and usedfor experiments between passages 3 and 10. EPCs were isolated from sevenhealthy newborns (three females and four males; gestational age range,38-40 weeks), pooled, expanded, and characterized according topreviously established protocol (Ingram et al., Blood, vol. 104, pp.2752-2760, 2004; Yoder et al., Blood, vol. 109, pp. 1801-1809, 2007;Mead et al., Curr. Protoc. Stem Cell Biol., vol. 2, p. 2C.1, 2008;Prater et al., Leukemia, vol. 21, pp. 1141-1149, 2007; Timmermans etal., J. Cell. Mol. Med., vol. 13, pp. 87-102, 2009). Briefly, EPCs wereexpanded in flasks coated with type I collagen (Roche Diagnostics,Basel, Switzerland), in endothelial growth medium (EGM; PromoCellHeidelberg, Germany) supplemented with 1 ng/ml VEGF₁₆₅ (Pierce,Rockford, Ill.), and incubated in a humidified incubator at 37° C. in anatmosphere containing 5% CO₂. EPCs were passaged every three to fourdays with 0.05% trypsin (Invitrogen, Carlsbad, Calif.) and characterizedfor the positive expression of cell-surface antigens CD31, CD141, CD105,CD144, vWF and Flk-1, and the negative expression of hematopoietic-cellsurface antigens CD45 and CD14. Single cell colony forming assays wereused to characterize their robust proliferative potential, secondary andtertiary colony formation upon replating.

Preparation of PEGDA Crosslinked Hyaluronic Acid (HA)-Gelatin Hydrogels.

HA-gelatin hydrogels (Extracel, Glycosan BioSystems, Inc., Salt LakeCity, Utah) were prepared as previously described (Vanderhooft et al.,Macromol. Biosci., vol. 9, pp. 20-28, 2009). Briefly, the hydrogels wereobtained by mixing 0.4% (w/v) Glycosil solution with 0.4% (w/v) Gelin-Ssolution in a 1:1 volume ratio with 1%, 0.4%, and 0.1% (w/v) ofpolyethylene glycol diacrylate (PEGDA) crosslinker (MW 3400) in a 4:1volume ratio, to obtain rigid, firm, and yielding substrates,respectively. The pregel solution was cast into a 96-well glass bottomplate (MatTek, Ashland, Mass.) for live/dead assay and CLSquantification and a 16-well Lab-Tek chamber slide (NUNC, Rochester,N.Y.) for TEM analysis and confocal images. Post-gelation, all hydrogelswere allowed to cure for 12 hours inside a biological safety cabinet tostabilize PEGDA-mediated crosslinking

Viscoelasticity Measurement.

Oscillatory shear measurements of the elastic modulus (G′) were obtainedusing a constant strain rheometer with steel cone-plate geometry (25 mmin diameter; RFS3, TA Instruments, New Castle, Del.) as previouslydescribed (Vanderhooft et al., Macromol. Biosci., vol. 9, pp. 20-28,2009). Briefly, oscillatory time sweeps were performed on three samples(n=3) for stiff, rigid, and yielding hydrogels to monitor the in situgelation. The strain was maintained at 20% during the time sweeps byadjusting the stress amplitude at a frequency of 1 Hz. This strain andfrequency were chosen because G′ was roughly frequency independentwithin the linear viscoelastic regime. The 24-hour tests occurred in ahumidified chamber at a constant temperature (25° C.) in 30 secondintervals. The Young's modulus (substrate stiffness) was calculated byE=2G′(1+v). HA-gelatin hydrogels can be assumed to be incompressible(Vanderhooft et al., Macromol. Biosci., vol. 9, pp. 20-28, 2009), suchthat their Poisson's ratios (v) approach 0.5 and the relationshipbecomes E=3G′ (Mammoto et al., Nature, vol. 457, pp. 1103-1108 2009;Boudou et al., Biorheology, vol. 43, pp. 721-728, 2006).

In a first stage of identifying suitably stiff hydrogels, differenthydrogels with the same HA:gelatin ratio, but with increased PEGDAcrosslinker concentration can be prepared to examine the gelationkinetics (FIG. 1A). Within 12 hours of HA-gelatin hydrogel curing,distinct Young's modulus (stiffness) profiles were established for rigid(650 Pa), firm (75 Pa), and yielding (10 Pa) HA-gelatin hydrogels. Afteran additional 12 hours of gelation, a slight increase in stiffness isobserved in all hydrogels (rigid to 780 Pa, firm to 85 Pa, and yieldingto 15 Pa). The 24 hour gelation period was sufficient to completelycrosslink all available thiol groups on the hydrogels at this HA:gelatinand PEGDA crosslinker composition (Vanderhooft et al., Macromol.Biosci., vol. 9, pp. 20-28, 2009). Indeed, further gelation time did notsignificantly increase substrate stiffness. Accordingly, HA-gelatinhydrogels cured for 12 hours, and used in the 12 to 24 hour periodprovide hydrogel mechanics that are fairly constant and can be used toevaluate EPC tube morphogenesis under various conditions.

Angiogenesis Assay.

Cord blood derived endothelial progenitor cells (CB EPCs) were seeded onrigid, firm, and yielding substrates, cured for 12 hours, with densitiesof 100,000 cells/cm². Constructs were cultured for 12 hours in EGM(PromoCell GmbH, Germany) supplemented with 1, 10, 25, or 50 ng/mlrecombinant human VEGF₁₆₅ (Pierce, Rockford, Ill.). Visualization andimage acquisition were performed using an inverted light microscope(Olympus IX50, Center Valley, Pa.) at time intervals of 3, 6, 9, and 12hours.

CB EPCs form functional and stable blood vessels in vivo compared toadult peripheral blood EPCs, which formed blood vessels that wereunstable and that regressed rapidly (Au et al., Blood, vol. 111, pp.1301-1305, 2008). EPCs have been used to study in vitro capillary tubeformation induced by substrate nanotopography (Bettinger et al., Adv.Mater., vol. 20, pp. 99-103, 2008). In vitro tube morphogenesis withlumen compartments was established as a prerequisite to define CB-EPCs(Timmermans et al., J. Cell. Mol. Med., vol. 13, pp. 87-102, 2009;Hirschi et al., Arterioscler. Thromb. Vasc. Biol., vol. 28, pp.1584-1595, 2008). To evaluate tube morphogenesis in a controllable invitro system, EPCs can be seeded on HA-gelatin hydrogel substrates curedfor 12 hours in media supplemented with either 1 ng/ml (low) or 50 ng/ml(high) VEGF. High concentrations of VEGF (i.e., 50 ng/ml) is known toinduce vascular differentiation of embryonic stem cells (Ferreira etal., Circ. Res., vol. 101, pp. 286-294, 2007) and vasculogenesis in HAhydrogels (Gerecht et al., Proc. Natl. Acad. Sci. U.S.A., vol. 104, pp.11298-11303, 2007). After 12 hours of incubation, no tube formation wasobserved in any culture supplemented with 1 ng/ml VEGF, while some CLSformed on the rigid and firm substrates, and predominant CLSs, similarto the CLSs observed on matrigel, formed on the yielding substratesupplemented with 50 ng/ml VEGF (FIG. 2A).

It should be noted that intermediate VEGF concentrations (e.g., 10 ng/mlor 25 ng/ml) increased tube morphogenesis on all substrates compared to1 ng/ml, but with inferior outcomes when compared to high VEGFconcentrations. We therefore continued our studies of tube morphogenesisusing high concentrations of VEGF.

Quantification of CLSs.

The LIVE/DEAD Viability/Cytotoxicity Kit (Invitrogen, Carlsbad, Calif.)was used to visualize CLSs, following the manufacturer's protocol.Briefly, calcein AM dye was diluted in phenol red-free DMEM (Invitrogen,Carlsbad, Calif.) to obtain a final concentration of 2 μM. Theconstructs were incubated with the dye solution for 30 minutes. Afterreplacing with fresh phenol red-free DMEM, CLSs were visualized using afluorescent microscope with a 10× objective lens (Axiovert, Carl ZeissInc., Thornwood, N.Y.). We analyzed four image fields per construct fromthree distinct experiments (n=3) performed in triplicate, usingMetamorph software 6.1 (Universal Imaging Co., Downingtown, Pa.) toquantify and compare CLSs formed on each substrate.

To study the kinetics of tube morphogenesis on substrates with differentmechanics, the following parameters were considered: (i) CLS phenotype;(ii) EPC layering and lengthening; and, (iii) cytoplasmic vacuoleformation, extension, and fusion to open lumen compartments. EPCs wereseeded on rigid, firm, and yielding substrates in media containing highVEGF concentrations. Time-interval images show that EPCs assembled in achain in an efficient and rapid manner on softer substrates (FIG. 8),resulting in a significant increase in tube length—from 130±3 μm onrigid substrate to 140±4 μm on firm substrate to 200±5 μm on yieldingsubstrate (FIG. 3A). Similarly, the area covered by CLSs alsoincreased—from 560±10 μm² on rigid substrate to 630±24 μm² on firmsubstrate to 1011±66 μm2 on yielding substrate (FIG. 3B). Although theextent of CLSs that formed on all of the substrates increased over time,with further incubation the extent of CLSs that formed on rigid and firmsubstrates did not achieve the predominance of CLSs that formed onyielding substrate (data not shown). In addition, CLSs that formed onyielding substrate were further found to be considerably thicker (20±1μm) than those that formed on either rigid (17±1 μm) or firm (18±0.3 μm)substrates, with branching and visible hollowing indicative of tubularstructure with open lumen space (FIG. 3C-3E).

Immunofluorescence.

EPCs cultured on HA hydrogels for 12 hours were fixed usingformalin-free fixative (Accustain, Sigma, St. Louis) for 20 minutes, andwashed with PBS. For staining, cells were permeabilized with a solutionof 0.1% Triton-X for ten minutes, washed with PBS, and incubated for onehour with mouse anti-human VE-Cad (1:200; BD Biosciences, San Jose,Calif.), rinsed twice with PBS, and incubated with anti-mouse IgG Cy3(1:50; Sigma, St. Louis). After rinsing twice with PBS, cells wereincubated with either FITC-conjugated lectin (1:40,Vector, Burlingame,Calif.) or FITCconjugated phalloidin (1:40, Molecular Probes, Eugene,Oreg.) for one hour, rinsed with PBS, and incubated with DAPI (1:1000;Roche Diagnostics, Basel, Switzerland) for an additional ten minutes.The hydrogels were gently placed into a glass bottom dish (Matek,Ashland, Mass.) and mounted with fluorescent mounting medium (Dako,Glostrup, Denmark). The immunolabeled cells were examined usingfluorescence microscopy (Olympus BX60, Center Valley, Pa.). A sequenceof z-stack images was obtained using confocal microscopy (LSM 510 Meta,Carl Zeiss Inc., Thornwood, N.Y.).

Transmission Electron Microscopy (TEM).

EPCs cultured on hydrogels for six and twelve hours were prepared forTEM samples as previously described (Perkins et al., Methods Mol. Biol.,vol. 372, pp. 467-483, 2007). Briefly, cells were fixed with 3.0%formaldehyde, 1.5% glutaraldehyde in 0.1 M Na cacodylate, 5 mM Ca²⁺, and2.5% sucrose at room temperature for one hour and washed three times in0.1 M cacodylate/2.5% sucrose pH 7.4 for 15 minutes each. The cells werepostfixed with Palade's OsO₄ on ice for one hour, rinsed withKellenberger's uranyl acetate, and then processed conventionally throughEpon embedding on a 16-well Lab-Tek chamber slide (NUNC, Rochester,N.Y.). Serial sections were cut, mounted onto copper grids, and viewedusing a Phillips EM 410 transmission electron microscope (FEI,Hillsboro, Oreg.). Images were captured with an FEI Eagle 2k camera.

To investigate the kinetics of cytoplasmic vesicle formation,coalescing, and elongation to an open lumen, TEM analysis was performedafter six and twelve hours of culture. After six hours, cytoplasmicvacuoles were observed in EPCs cultured on all substrates. However,while EPCs on rigid substrate were organized into two to three celllayers, they elongated on firm substrate and both elongated andorganized into a single layer on the yielding substrate (FIGS. 4A-4C).After twelve hours of culturing, EPCs on rigid substrate were moreelongated and contained many vacuoles. On firm substrate, cells wereelongated with larger vacuoles, and lumen compartments were observed onseveral occasions. On yielding substrate, we observed complex structureswith open lumen spaces and very lengthened cells (FIGS. 4D-4F).

RNAi transfection:

EPCs were transfected with siGENOME SMARTpool human Cdc42 and MT1-MMP(Dharmacon, Lafayette, Colo.) using the manufacturer's protocol.Briefly, the RNAi transfection solution was prepared by mixing aserum-free and antibiotic-free EGM (PromoCell GmbH, Germany) withDharmaFECT2 RNAi transfection reagent (Dharmacon). EPCs were cultured to90% confluence on a 24-well plate (NUNC, Roskilde, Denmark). Fortransfection, EPC growth medium was removed and replaced with 400 μL, ofantibiotic-free EGM (PromoCell, Heidelberg, Germany) and 100 μL,transfection solution in each well, to achieve a final RNAiconcentration of 50 nmol/L. Transfected cells were incubated at 37° C.,and the medium was replaced with a fresh EGM medium after 24 hours. RNAanalysis was performed after 48 hours, and protein analysis wasperformed after 72 hours. Confirmed transfected EPCs were used forexperiments after 48 to 96 hours.

MT1-MMP has been reported to support neovascularization by allowingmatrix degradation at the migrating cell front (Van Hinsbergh et al.,Arterioscler. Thromb. Vasc. Biol., vol. 26, pp. 716-728, 2006), as wellas by creating a physical space to control tube and lumen morphogenesis(Chun et al., J. Cell. Biol., vol. 167, pp. 757-767, 2004; Saunders etal., J. Cell Biol., vol. 175, pp. 179-191, 2006). The function ofMT1-MMP in tube morphogenesis of EPCs on various levels of substratestiffness was examined using an RNAi suppression approach. EPCs treatedwith siRNA targeting the MT1-MMP (FIG. 9A) were seeded on the differentsubstrates. In contrast to the Luciferase-treated EPCs (control), siRNAsuppression of MT1-MMP mitigated CLS formation on firm and yieldingsubstrates, with more rounded cell morphology, while allowing cellspreading on rigid substrates (FIGS. 5A, 5C, 5E and 9E).

Cdc42 has been demonstrated to regulate EC morphogenesis through vacuoleand lumen formation (Bayless et al., J. Cell. Sci., vol. 115, pp.1123-1136, 2002), as well as to mediate cell spreading, motility,growth, and differentiation through cytoskeletal remodeling and focaladhesion assembly (Jaffe et al., Annu. Rev. Cell Dev. Biol., vol. 21,pp. 247-269, 2005; Mammoto et al., Curr. Opin. Hematol., vol. 15, pp.228-234 2008). An RNAi suppression approach was used to examine whetherCdc42 is the connecting link by which EPCs respond to substratestiffness during tube morphogenesis. EPCs were treated with siRNA thattargeted the small Rho GTPase Cdc42 (FIG. 9B) and were seeded on thedifferent substrates. Luciferase-treated (control) EPCs were able tospread and form some extent of CLSs on rigid and firm substrates andcould form CLSs to a greater extent on yielding substrate. However,siRNA suppression of Cdc42 prevented EPC tube morphogenesis on allsubstrates; we observed rounded cell morphology on the yieldingsubstrate and a more spreading morphology on the rigid and firmsubstrates (FIGS. 5B, 5D, and 5F and FIG. 9E).

Real-Time RT-PCR.

Two-step RT-PCR was performed on EPCs cultured in media supplementedwith 1 ng/ml or 50 ng/ml VEGF. Total RNA was extracted using TRIzol(Gibco, Invitrogen Co., Carlsbad, Calif.) according to themanufacturer's instructions. Total RNA was quantified by an ultravioletspectrophotometer, and the samples were validated for no DNAcontamination. RNA (1 μg per sample) was reversed transcribed usingM-MLV (Promega Co., Madison, Wis.) and oligo(dT) primers (Promega Co.)according to the manufacturer's instructions. We used the TaqManUniversal PCR Master Mix and Gene Expression Assay (Applied Biosystems,Foster City, Calif.) for MMP-1, MMP-2, MT1-MMP, HPRT1 and β-ACTIN,according to the manufacturer's instructions. The TaqMan PCR step wasperformed in triplicate with an Applied Biosystems StepOne Real-Time PCRsystem (Applied Biosystems), using the manufacturer's instructions. Therelative expression of MMP1, MMP2, and MT 1-MMP was normalized to theamount of HPRT1 or β-ACTIN in the same cDNA by using the standard curvemethod described by the manufacturer. For each primer set, thecomparative CT method (Applied Biosystems) was used to calculateamplification differences between the different samples. The values forexperiments (n=3) were averaged and graphed with standard deviations.Similar procedures were used to analyze the expressions of Cdc42 andMT1-MMP from the RNAi experiments.

To examine whether VEGF would induce MMP production, real time RT-PCRwas performed to compare MMP expression in EPCs cultured on rigid, firm,and yielding substrates supplemented with high VEGF, to theircounterparts cultured with low VEGF. After 12 hours of incubation inmedia supplemented with high VEGF, EPCs cultured on all substrate showedincreased production of MMPs. Specifically, EPCs cultured on rigid andfirm substrates with high VEGF produced three times the MMP-2 and fourtimes the MMP-1 and MT1-MMP produced by EPCs cultured in mediasupplemented with low VEGF concentration. EPCs cultured on yieldingsubstrate supplemented with high VEGF showed a smaller, but significant,increase in MT1-MMP, MMP-1, and MMP-2 compared to their counterpartcultured in low VEGF concentration (FIGS. 2B-2D). When EPCs werecultured in media supplemented with high VEGF concentration, the MMPproduction decreases as the stiffness of the substrate was reduced(FIGS. 2E-2G).

Western Blot.

The relative amounts of MT1-MMP or Cdc42 in RNAi transfected EPCs weredetected using Western Blotting of whole cell lysates prepared instandard extraction buffer containing 4% SDS, 20% glycerine, and 0.0125M Tri-HCl pH 6.8. Protein (20 μg) from these samples was separated in a12.5% Criterion Tris-HCl Gel (Bio-Rad Lab., Hercules, Calif.) for 40minutes using Tris/glycine/SDS running buffer. Resolved proteins weretransferred to immuno-blot nitrocellulose membranes (Bio-Rad Lab) andblocked with 3% dry-milk in Tris buffered salinetween (TBS-T). Themembranes were immunoprobed overnight with anti-human rabbit monoclonalantibodies to MT1-MMP (1:1,000, Epitomics, Burllingame, Calif.) and toCdc42 (1:1,000, Cell Signaling Technology, Beverly, Mass.). For loadingcontrol, the membranes were probed with anti-human mouse beta-Actin(1:1,000, Cell Signaling Technology). Next, membranes were treated witheither HRP-conjugated anti-rabbit or anti-mouse IgG secondary antibodies(1:1,000, Cell Signaling Technology) and then developed by addingSuperSignal West Pico Chemiluminescent Substrate (Thermo ScientificPierce, Rockford, Ill.).

Statistical Analysis.

Statistical analysis of CLSs quantification, MMP production, and RNAisuppression data was performed using GraphPad Prism 4.02 (GraphPadSoftware Inc., La Jolla, Calif.). Parametric two way ANOVA test wereperformed to assess the significance of MMP production by EPCs amongrigid, firm, and yielding substrates cultured in low and high VEGFconcentrations. Unpaired Student's t-tests were performed to analyzeCLSs quantification and RNAi suppression data. Significance levels wereset at *p<0.05, **p<0.01, and ***p<0.001, respectively.

Results

Hydrogels with defined compositions and tunable elasticity can be usedfor in vitro tube morphogenesis. These hydrogels served as substrates ofvarying stifihesses, allowing for varying the kinetics of EPCtubulogenesis. Viscoelasticity measurements during in situ gelationdemonstrate three distinct substrate stiffness profiles: rigid, firm,and yielding. While low levels of VEGF allowed EPCs to spread on allsubstrates, high levels of VEGF were required to initiate tubemorphogenesis and to activate MMPs, whose expression declined with thedecrease of matrix stiffness. We then observed that increased tubemorphogenesis—including CLS progression, vesicle formation, expansion(in both size and number) and fusion to lumens—corresponded to decreasedsubstrate rigidity. RNA interference (RNAi) studies further showed thatEPCs required MT1-MMP and Cdc42 to undergo tube morphogenesis.

Utilizing chemically and mechanically defined hydrogels in an in vitroculture system for angiogenesis, tube morphogenesis can be evaluatedover a range of elasticity softer than usually available in vitro andrelevant for vascular morphogenesis (10 Pa to 650 Pa), as well as theresponse to growth factor administration. Growth factors, for exampleVEGF, are a prerequisite for instigation of angiogenesis and laterprogress in tube morphogenesis regulated by mechanical stresses from theECM (FIG. 6A). First, MMP production induced by growth factors reducesthe mechanical resistance of the ECM and allows EPC migration andreorganization. This MMP production is co-regulated by matrix stiffness,as EPCs cultured on the rigid and firm substrates have to produce moreMMPs to overcome the extra mechanical barrier. At this stage, reducedelasticity of the ECM promotes tubulogenesis, which is characterized bya significant increase in mean tube length, tube area, and thickness asmatrix stiffness is reduced from rigid to yielding. Although EPCscultured on rigid and firm substrates produce more MMPs, most likely toovercome the extra mechanical resistance, the local decrease insubstrate stiffness cannot support predominant CLS formation. Hence,EPCs cultured on the yielding substrates are able to form predominantCLSs with extended vacuoles and open lumens.

Vessel morphogenesis is a highly dynamic process in which invasion,motility, and lumenogenesis occur concurrently in different regions ofthe developing tube. In contrast to angiogenic sprouting, cell hollowingis a mechanism by which individual cells generate vacuoles through thepinocytosis process; these vacuoles then coalesce, forming a lumen thatconnects to the lumens of neighboring cells (Lubarsky et al., Cell, vol.112, pp. 19-28, 2003; Kamei et al., Nature, vol. 442, pp. 453-456, 2006;Davis et al., Exp. Cell. Res., vol. 224, pp 39-51, 1996. Iruela-Arispeet al., Dev Cell., vol. 16, pp. 222-231, 2009). In response to VEGF andmatrix stiffness, EPCs produce MMPs that allow further cellularmigration and connection into CLSs (FIG. 2A). Furthermore, asdemonstrated by the kinetics of CLS formation along 12 hours (FIG. 8),vacuoles formation occurs within 6 hours (FIGS. 4A-4C) and enlargedvacuoles and lumens within 12 hours (FIGS. 4D-4F).

Example 2 Materials and Methods Human ECFCs

Human umbilical cord blood ECFCs isolated from outgrowth clones,provided by Dr. Yoder, Indiana University School of Medicine, expandedin endothelial growth media (EGM; PromoCell GmbH, Germany) and used forexperiments between passages 6 and 8 as previously described(Hanjaya-Putra et al., Journal of Cellular and Molecular Medicine, vol.14, no. 10, pp. 2436-2447, 2010).

Synthesis of AHA-Hydrogels

Acrylated hyaluronic acid (AHA) was synthesized using a two-stepprotocol: (1) Synthesis of the tetrabutylammonium salt of HA (HA-TBA)was performed by reacting sodium hyaluronate (64 kDa, Lifecore) with thehighly acidic ion exchange resin Dowex-100 and neutralization with 0.2 MTBA-OH. (2) Coupling of acrylic acid (2.5 eq) and HA-TBA (1 eq, repeatunit) in the presence of dimethylamino pyridine (DMAP; 0.075 eq) anddi-tert-butyl-dicarbonate (1.5 eq) in DMSO, followed by dialysis andlyophilization. 1H NMR spectrum was used to confirm the final percentmodification of the AHA-hydrogels.

Peptides

The cell adhesive peptide GCGYGRGDSPG (MW: 1025.1 Da; bold italicsindicates RGD integrin-binding domain), cell non-adhesive peptideGCGYGRDGSPG (MW: 1025.1 Da; bold italics indicates RDG the mutatedintegrin-binding domain), MMP-sensitive crosslinker GCRDGPQG↓IWGQDRCG(MW: 1754.0 Da; down arrow indicate the site of preteolytic cleavage),and MMP-insensitive crosslinker GCRDGDQGIAGFDRCG (MW: 1754.0 Da), allwith >95% purity (per manufacturer HPLC analysis), were obtained fromGenScript Corporation (Piscataway, N.J., USA).

Generation of Vascular Constructs.

AHA polymer (3 wt %) was dissolved in a triethanolamine-buffered saline(TEOA buffer: 0.2 M TEOA, 0.3 M total osmolarity, pH 8.0). The cellsadhesive peptides (RGD, genscript, N.J.) dissolved in TEOA buffer wasadded to the AHA solution at a final peptide concentration of 0.37 mM,3.7 mM, and 14.8 mM (corresponding to 1%, 10%, and 20% of availableacrylate groups within 3 wt % AHA). Recombinant human VEGF₁₆₅ (Pierce,Rockford, IL), bFGF (Invitrogen), Ang-1 (R&D), TNF-α, and SDF-1 wereadded at 50 ng/ml into the AHA-RGDS mixture and allowed to react for 1hour with gentle shaking Human umbilical cord blood ECFCs wereencapsulated in HA hydrogels with densities of 5.10⁶ cells/ml. Followingthe re-suspension of cells into this solution, MMP peptide crosslinker(MMP, genescript) dissolved in TEOA buffer was added (corresponding tothe 20% of available acrylate groups within 3 wt % AHA). Immediatelyafter the addition of the MMP crosslinker, 50 μl of this mixture waspipetted into sterile molds (5 mm diameter, 2 mm height), and allowed toreact for 15 minutes at room temperature inside the laminar flow hood.The formed constructs were cultured for up to 3 days in EGM (PromoCellGmbH, Germany). Visualization and image acquisition were performed usingan inverted light microscope (Olympus IX50, Center Valley, Pa.) andconfocal microscope (LSM 510 Meta, Carl Zeiss Inc., Thornwood, N.Y.) atvarious time points.

Viscoelasticity Measurement.

Oscillatory shear measurements of the elastic modulus (G′) were obtainedusing a constant strain rheometer with steel cone-plate geometry (25 mmin diameter; RFS3, TA Instruments, New Castle, Del.) as previouslydescribed (Hanjaya-Putra et al., Journal of Cellular and MolecularMedicine, vol. 14, no. 10, pp. 2436-2447, 2010; Vanderhooft et al.,Macromolecular Bioscience, vol. 9, pp. 20-28, 2009). Briefly,oscillatory time sweeps were performed on three samples (n=3) for eachhydrogels samples at various time point. The strain was maintained at20% during the time sweeps by adjusting the stress amplitude at afrequency of 1 Hz. This strain and frequency were chosen because G′ wasroughly frequency independent within the linear viscoelastic regime. Thetests occurred in a humidified chamber at a constant temperature (25°C.) in 30 second intervals. The Young's modulus (substrateviscoelasticity) was calculated by E=2G′(1+v). HA-gelatin hydrogels canbe assumed to be incompressible (Vanderhooft et al., MacromolecularBioscience, vol. 9, pp. 20-28, 2009), such that their Poisson's ratios(v) approach 0.5 and the relationship becomes E=3G′ (Mammoto et al.,Nature, vol. 457, pp. 1103-1108, 2009, Boudou et al., Biorheology, vol.43, pp. 721-728, 2006).

VEGF Released and Degradation Study

At various time points, 1 mL of conditioned media from the gels aloneand gels containing cells were collected and replaced with a freshgrowth media. At final time point (day 3), the gels were degraded usingendogenous 1,000 IU/ml hyaluronidase IV (Sigma, XXX). After 24 hours thegels were completely degraded and the conditioned media were collected.All of the samples (n=3) were stored at −80° C. before performing anELISA analysis for VEGF and uronic acid assay. VEGF release profile wereperformed using an ELISA kit (Pierce Biotechnology) followingmanufacturer's instructions. Briefly, VEGF₁₆₅ in the ELBA kit standardsand samples were captured on the anti-human VEGF165 antibody-coatedmicroplate. After removing unbound proteins, biotinylated antibodyreagent was added to bind to the secondary site on VEGF₁₆₅. Then, toproduce a colorimetric signal, streptavidin-horseradish peroxidase wasadded to bind to TMB. Standards were prepared according to themanufacturer's instructions. Plate washing was performed three timesbetween each step to remove any excess reagents. The colorimetric signalwas detected using a UV microplate spectrophotometer (SpectraMax Plus,Molecular Devices, Sunnycale, Calif.) at absorbance wavelengths of 450and 550 nm. The standard curve was interpolated to determine the amountof VEGF165 at each predetermined time point. Results are presented interms of cumulative release as a function of time. To study thedegradation kinetic of the gels by the cells, the conditioned media wasalso analyzed via a modified uronic acid assay as previously reported(Burdick et al., Biomacromolecules, vol. 6, no. 386-391, 2005).

Vacuoles Visualization and Integrins Blocking

Quantitation of vacuoles and lumen formation was performed followingprevious reported protocol (Bayless et al., Am. J. Pathol., vol. 156,pp. 1673-1683, 2000). For each condition 200 cells were analyzed forvacuole and lumen formation. A cell was considered to be vacuolating ifmore than 30% of the cell's area contained a vacuole or lumen. Forfurther visualization using confocal microscopy, FM-464 vacuole staining(Invitrogen) was performed following manufacturer's protocol. Todetermine the integrins involved, various antibodies directed towardhuman integrin subunits and heterodimers were added.

Immunofluorescence.

Human umbilical cord blood ECFCs cultured in flasks or encapsulatedECFCs cultured within HA hydrogels for 48 hours were fixed usingformalin-free fixative (Accustain, Sigma) for 20 minutes, and washedwith PBS. For staining, cells were permeabilized with a solution of 0.1%Triton-X for ten minutes, washed with PBS and incubated for one hourwith mouse anti-human VE-Cad (1:200; BD Biosciences, San Jose, Calif.),anti-human CD44 (1:100; Sigma), or anti-human CD168 (1:50; Novocastra,Newcastle, UK) rinsed twice with PBS, and incubated with anti-mouse IgGCy3 (1:50; Sigma). After rinsing twice with PBS, cells were incubatedwith either FITC-conjugated lectin (1:40,Vector, Burlingame, Calif.) orFITC-conjugated phalloidin (1:40, Molecular Probes, Eugene, Oreg.) forone hour, rinsed with PBS, and incubated with DAPI (1:1000; RocheDiagnostics, Basel, Switzerland) for an additional ten minutes. Thehydrogels were directly imaged by inverted fluorescence microscopy inthe 96-well glass bottom plate. The immunolabeled cells were examinedusing using confocal microscopy (LSM 510 Meta, Carl Zeiss Inc.).

Transmission Electron Microscopy (TEM).

At various time points throughout vascular morphogenesis, the ECFCs andHA-hydrogels constructs (n=3, for each time point) were prepared for TEMsamples as previously described (Perkins et al., Methods Mol. Biol.,vol. 372, pp. 467-483, 2007). Briefly, the constructs were fixed with3.0% formaldehyde, 1.5% glutaraldehyde in 0.1 M Na cacodylate, 5 mMCa2+, and 2.5% sucrose at room temperature for one hour and washed threetimes in 0.1 M cacodylate/2.5% sucrose pH 7.4 for 15 minutes each. Thecells were postfixed with Palade's OsO₄ on ice for one hour, rinsed withKellenberger's uranyl acetate, and then processed conventionally throughEpon embedding. Serial sections were cut, mounted onto copper grids, andviewed using a Phillips EM 410 transmission electron microscope (FEI,Hillsboro, Oreg.). Images were captured with an FEI Eagle 2k camera.

Scanning Electron Microscopy (SEM).

The ultrastructure of the hydrogels was studies using SEM (FEI QuantaESEM 200) as was previously described (Sun et al., CarbohydratePolymers, vol. 65, pp. 273-287, 2006). Briefly, the hydrogels wereswelled in PBS for 24 hours, quickly frozen in liquid nitrogen, and thenfreeze-dried in a Virtis Freeze Drier (Gardiner, N.Y.) under vascuum at−50° C. for 3 days until the samples became completely dry. Thefreeze-dried hydrogels were fractured carefully to reveal the interiorand mounted onto aluminum stubs with double side carbon tape andsputter-coated (Anatech Hummer 6.2 Sputter Coater) with Ag for 1 minute.The interior morphology of the hydrogels was examined using SEM at 25 kVand 12 nA.

Quantification of CLSs.

The LIVE/DEAD Viability/Cytotoxicity Kit (Invitrogen, Carlsbad, Calif.)was used to visualize CLSs, following the manufacturer's protocol.Briefly, calcein AM dye was diluted in phenol red-free DMEM (Invitrogen,Carlsbad, Calif.) to obtain a final concentration of 2 μM. Theconstructs were incubated with the dye solution for 30 minutes. Afterreplacing with fresh phenol red-free DMEM, CLSs were visualized using afluorescent microscope with a 10× objective lens (Axiovert, Carl ZeissInc.). We analyzed four image fields per construct from three distinctexperiments (n=3) performed in triplicate, using Metamorph software 6.1(Universal Imaging Co., Downingtown, Pa.) to quantify and compare CLSsformed within each hydrogel.

Real-Time RT-PCR.

Two-step RT-PCR was performed on ECFCs cultured in media supplementedwith 1 ng/ml or 50 ng/ml VEGF. The human colon carcinoma cell lineLS174T (ATCC, Manassas, Va.) was used as positive control. Total RNA wasextracted using TRIzol (Gibco, Invitrogen Co., Carlsbad, Calif.)according to the manufacturer's instructions. Total RNA was quantifiedby an ultraviolet spectrophotometer, and the samples were validated forno DNA contamination. RNA (1 μg per sample) was reversed transcribedusing M-MLV (Promega Co., Madison, Wis.) and oligo(dT) primers (PromegaCo.) according to the manufacturer's instructions. We used the TaqManUniversal PCR Master Mix and Gene Expression Assay (Applied Biosystems,Foster City, Calif.) for SPAM1, HPRT1 and β-ACTIN, according to themanufacturer's instructions. The TaqMan PCR step was performed intriplicate with an Applied Biosystems StepOne Real-Time PCR system(Applied Biosystems), using the manufacturer's instructions. Therelative expression of SPAM1 was normalized to the amount of HPRT1 orβ-ACTIN in the same cDNA by using the standard curve method described bythe manufacturer. For each primer set, the comparative CT method(Applied Biosystems) was used to calculate amplification differencesbetween the different samples. The values for experiments (n=3) wereaveraged and graphed with standard deviations.

Chick Embryo Chorioallantoic Membrane (CAM) Assay

Chick embryo chorioallantoic membrane (CAM) was cultured ex ovo.Briefly, fertilized chicken eggs (Berries farm, P.A.) were incubated at37° C. and 60% humidity for 3-4 days. At this time point (E.3-4), theeggs were cleaned with betadine and 70% ethanol and cracked opened intoa petri dish, which contained 5-10 mL of DMEM media (invitrogen). TheCAM was cultured ex ovo in a humidified incubator at 37° C. in anatmosphere containing 5% CO₂. The vascularized constructs (n=8-10, oneconstruct per CAM) were cultured in vitro for 3 days and grafted ontoCAM membrane at E.8, and were cultured for an additional 4 days (E.12),in which period the host EC mitotic index is optimum. At E.12,FITC-lectin or dextran was perfused into a major aorta and theconstructs were harvested and fixed in formalin free Accustain fixative(Sigma). Analysis was performed using both confocal microscopy andhistological sectioning.

Transplantation of Vascularized Constructs

Vascularized constructs were implanted into the flanks of 6-8 weeks oldNUDE mice following well established protocol by Dr. Yoder (Mead et al.,Current protocols in stem cell biology, vol. 6, pp. 2C.1.1-2C.1.27,2008). Briefly, vascularized constructs were cultured in vitro for 3days and implanted into each flanks (left or right, 2 constructs permouse). Eight to ten constructs were implanted for each groups (n=8-10).After two weeks, mice were euthanized and the constructs were harvestedand fixed in formalin free Accustain fixative (Sigma). Explants werethen paraffin embedded, bisected along major axis, and sectioned (5 μmthick) for immunohistochemical analysis. All animal protocols wereapproved by the Johns Hopkins University Institutional Animal Care andUse Committee.

Statistical Analysis.

Analysis of CLSs quantification and hyaluronidase expression data wasperformed on triplicate samples with quadruplicate and duplicatereadings at each data point, respectively, and statistical analysis wasperformed using GraphPad Prism 4.02 (GraphPad Software Inc.). T-testswere performed to determine significance using GraphPad Prism 4.02(GraphPad Software Inc.). Significance levels were determined usingposttests among the three substrates, and were set at *p<0.05, **p<0.01,and ***p<0.001, respectively. All graphical data were reported.

Results

Acrylated HA (AHA) can provide the dynamic cues required for networkassembly of encapsulated ECFCs (FIG. 10A). These hydrogels are formedusing sequential crosslinking from acrylated HA precursors by reactingwith thiol-terminated RGD integrin-binding peptide (thiol group on RGDSreact with acrylates along the HA backbone) followed by an additionreaction with thiol-terminated peptide crosslinkers (thiol groups oneach end of peptide react with acrylates along the HA backbone) (Khetanet al., Soft Matter, vol. 5, no. 8, pp. 1601-1606, 2009). In these AHAhydrogels, an enzymatically degradable peptide that can be cleaved byboth MMP-1 and MMP-2 (Lutolf et al., Proc. Natl. Acad. Sc U.S.A., vol.100, no. 9, pp. 5413-5418, 2003; Seliktar et al., J. Biomed. Mater. Res.A, vol. 68, no. 4, pp. 704-716, 2004), for example a peptide with thesequence GCRDGPQGJ↓IWGQDRCG, can be used as a crosslinker. An adhesiveligand, such as RGD, which is the sequence within the fibronectinmolecule that mediates cell attachment, can also be included. The AHAhydrogels can have about 50% modification (50% of the repeating HAmacromer contains acrylate group) to form 3 wt % hydrogels (FIGS. 10Band 10C). Higher acrylate modification and wt % corresponds torelatively dense networks can also be used but do not as effectivelysupport vascular morphogenesis.

Adhesion Compounds

The incorporation rate of the RGD containing peptide can be altered inAHA hydrogels (Khetan et al., J. Vis. Exp., (32), 2009; Khetan et al.,Conf. Proc. IEEE Eng. Med. Biol. Soc., vol. 1, pp. 2094-2096, 2009) todetermine its effect on tubulogenesis. Based on previous publicationsexamining the role of RGD adhesion site in tubulogenesis in fibrin gels(Bayless et al., Am. J. Pathol., vol. 156, no. 5, pp. 1673-1683, 2000),AHA hydrogels were generated with RGD concentrations of 0.37, 3.7, and14.8 mM, and the kinetics of vacuole and lumen formation were analyzedat various time intervals. Vacuole and lumen formation is dependent onadhesion molecule e.g. RGD, concentration and an example of an optimalRGD concentration in our system is 3.7mM, which corresponds to 10% ofacrylate consumption (FIG. 11A). Very little vacuole formation with nolumen is detected in AHA hydrogels generated with 0.37 mM RGD. Higherconcentrations of RGD did not significantly enhanced vacuole formationbut created viscous gels which were difficult to image and handle.

When a mutated adhesion site, RGE, was used, little to no vacuole andlumen formation is observed. These results are in agreement withprevious published data that RGD regulates vacuoles and lumen formation(Bayless et al., Am. J. Pathol., vol. 156, no. 5, pp. 1673-1683, 2000),and that optimum concentration of RGD similar to fibrin gels can be usedto initiate vascular morphogenesis in synthetic hydrogels (Gobin et al.,FASEB J.: 01-0759fje; Lutolf et al., Nature Biotechnology, vol. 23, no.1, pp. 47-55, 2005; Raeber et al., Biophysical Journal, vo. 89, no. 2,pp. 1374-1388, 2005).

Crosslinker

MMP-sensitive crosslinker incorporation into AHA hydrogels (See FIG.11C) can be used. When RGE and MMP-insensitive crosslinker are used, novacuole or lumen is observed. However, when RGD and MMP-insensitivecrosslinker are used, some vacuoles with limited extent of lumencoalescence are observed. This suggest that RGD not only regulatesvacuoles formation but also may localized MMPs activation on the cellsurface (Xu et al., The Journal of Cell Biology, vol. 154, no. 5, pp.1069-1080, 2001), enabling cell migration and coalescence into lumenalnetwork.

These results show that RGD regulate vacuoles and lumen formation withinsynthetic AHA hydrogels in a dose dependent manner. Once vacuoles areformed, ECFCs migrate to the nearest neighbor to further coalescenceinto lumen structure. Like any other cell types, ECFCs migration within3D matrix requires multiple balances between integrin activity, RGDligand density, and proeteolytic activity (Zaman et al., Proceedings ofthe National Academy of Sciences, vol. 103, no. 29, pp. 10889-10894,2006). Medium RGD density which allow optimum cell migration (Gobin etal., FASEB J.: 01-0759fje), might be conducive for vascularmorphogenesis to precede forward into lumen formation and networkbranching. These results are in agreement with what were observed in 2Dculture when ECs were cultured on surface coated with fibronectin. Inmedium ECM density, with the appropriate RGD adhesion peptide, ECscollectively retracted and formed into branching capillary networks withhollow tubular structure (Ingber et al., J. Cell. Biol., vol. 109, pp.317-330, 1989).

Both MMP and adhesion molecules such as RGD are required for ECFCstubulogenesis in AHA gels. Matrix parameters and their involvement inother signaling pathways during tubulogenesis can be decoupled todevelop further gel characteristics.

Vascular Network Assembly of ECFC within AHA Gels

Encapsulation and Morphogenesis

Throughout the RGD and MMP experiments described above, vacuoleformation is completed, reaching an optimum within 48 hours ofencapsulation, which suggests that vacuole and lumen formation iscompleted at the end of day 1. Vacuoles can be observed within 3-6 hoursafter cell encapsulation at day 0 (FIGS. 12A-12D). This processcontinues with increased number and size of vacuoles, following withcoalescence into larger lumen (FIGS. 12E-12H).

Branching and Sprouting

On the second day of culture, progression of tubulogenesis was observedthrough both branching and sprouting, with clear AHA hydrogeldegradation at the cell microenvironment, changes in cell morphologytoward elongation, and guiding channels forming between neighboringcells (FIGS. 13A-13H).

Network Formation and Growth

By day 3, vascular networks grow, and complex and comprehensivestructures are clearly observed within AHA hydrogels (FIG. 14A-14F).Open lumen within the networks is easily detected throughout thehydrogel (FIGS. 14D and 14E)

Cell and Material Interaction: Kinetics of Matrix Properties alongVascular Morphogenesis.

The dynamic interactions between ECFCs and AHA hydrogels were alsocharacterized. AHA hydrogel stiffness decreases along the progressionand network formation, reaching 40 Pa, which are in similar ranges tothe experiments described above for HA-gelatin hydrogels (See alsoHanjaya-Putra et al., Journal of Cellular and Molecular Medicine, vol.14, no. 10, pp. 2436-2447, 2010). This further demonstrates thathydrogel stiffness should be controlled to enable in vitro vascularmorphogenesis (FIG. 15A). The observation that enhanced vascularnetworks are formed as matrix stiffness is decreased, is consistent withresults of previous studies using self-assembling peptide (Sieminski etal., Cell Biochem. Biophys., vol. 49, pp. 73-83, 2007), matrigel(Deroanne et al., Cardiovasc. Res., vol. 49, pp. 647-658, 2001),collagen (Sieminski et al., Exp. Cell. Res., vol. 297, pp. 574-584,2004), and fibrin gels (Stephanou et al., Microvascular Research, vol.73, no. 3, pp. 182-190, 2007; Kniazeva et al., Am. J. Physiol. CellPhysiol., vol. 297, no. 1, pp. C179-187, 2009).

The optimal magnitude of matrix stiffness for different gel types is abit different, but this can be attributed to the specificity ofdifferent gels, a universal nature of this trend in various gel typesemphasizes that cellular remodeling is importance for vascularmorphogenesis. Therefore, a SEM study was done to further investigatecellular remodeling of ECFCs within AHA hydrogels. SEM images of day 1show rounded cells within complex hydrogel mesh ultrastructure, which byday 3 are spread in between large guiding microchannels (FIG. 15C).These guiding microchannels are formed by MMPs and hyaluronidases, whichexpression is increased by ECFCs along the 3-day culture period (FIG.15D). Previous studies in 3D collagen gels have shown that ECs create“vascular guidance tunnel,” to allow cells migration and vascularnetworks formation. Within these physical spaces ECs move in MT1-MMPdependent manner to form complex lumen structures (Sacharidou et al.,Blood, vol. 115, no. 25, pp. 5259-5269, 2010; Stratman et al., Blood,vol. 114, no. 2, pp. 237-47, 2009).

Additional Growth Factors

SDF-1 and TNF-alpha are known to induce MMP production in ECs (Han, Tuanet al. 2001), in which an optimum MMP secretion was needed to allowvascular branching and network formation (Sacharidou et al., Blood, vol.115, no. 25, pp. 5259-5269, 2010; Iruela-Arispe et al., Dev. Cell, vol.16, pp. 222-231, 2009; Stratman et al., Blood, vol. 114, no. 2, pp.237-47, 2009). In hydrogels of the present invention, encapsulated SDF-1and TNF-alpha can be used to induce MMP production to allow sproutingand invasion, as was reported in other synthetic hydrogels (Raeber etal., Biophysical Journal, vo. 89, no. 2, pp. 1374-1388, 2005).

Monitoring

The kinetics of cellular remodeling can be observed at various pointsthroughout vascular morphogenesis by monitoring the uronic acid, abyproduct of the AHA degradation, released in the media. As vascularmorphogenesis progresses, ECFCs secrete MMP-1,-2, MT1-MMP, Hyal-2, andHyal-3, the inventive AHA hydrogels are degraded and as a result matrixstiffness decreases.

During this process, AHA hydrogel was degraded and released the uronicacid byproduct to the culture media (FIG. 15F), enabling progression intubulogenesis. As the hydrogels were degraded, about 80% of theencapsulated VEGF is released into the media providing a soluble cue forthe ECFCs to undergo tubulogenesis (FIG. 15E). These data demonstratethat a suitable synthetic material provides a responsive and dynamicniche to accommodate vascular morphogenesis, through activation ofproteases specific for the tubulogenesis microenvironment (Lutolf etal., Nature Biotechnology, vol. 23, no. 1, pp. 47-55, 2005; Stratman etal., Blood, vol. 114, no. 2, pp. 237-47, 2009).

Molecular Regulation of Vascular Morphogenesis in AHA Gels.

To determine the integrin subunit that regulates vacuole and lumenformation within this synthetic AHA hydrogels, integrin blockingexperiment was performed. Three integrins αVβ3, α5β1, α2β1 are known toregulate vacuole and lumen formation in collagen and fibrin gels(Bayless et al., Am. J. Pathol., vol. 156, no. 5, pp. 1673-1683, 2000).Blocking α2β1 integrin did not significantly reduced vacuole and lumenformation compared to control (FIG. 16A). However, blocking α5β1integrin reduced the extent of vacuole and lumen formation to 20%.Moreover, blocking αvβ3 significantly blocked vacuole and lumenformation down to 10% after 48 hours. Indeed, αv is found to be highlyexpressed on day 0, while decreasing along the cultures period; α5upregulate on day 1 of culture; whereas the expression of both β1 and β3is decreased on days 2 and 3 of the culture period (FIG. 16B).Altogether, in the synthetic culture system, integrin subunits α5, αV,β1 and β3 are highly expressed at early stage of vascular morphogenesis(day 0-day 1), to initiate vacuole and lumen formation throughRGD-integrin signaling cascade. This phenomena was reported in fibringels (Bayless et al., Am. J. Pathol., vol. 156, no. 5, pp. 1673-1683,2000) where RGD induce vacuole and lumen formation through α5β1 andαVβ3, the main RGD-binding integrins (Xu et al., The Journal of CellBiology, vol. 154, no. 5, pp. 1069-1080, 2001). The integrin expressionis offset by the increase in MMPs expression to support branching andnetwork formation (day 2-day 3) (FIG. 16D). Specifically, both MMP-1 andMMP-2 are localized near the cell membrane at day 2, where branching andsprouting start to take place (FIG. 16C). In collagen and fibrin gels,TIMPs expressed by ECs and pericytes were shown to stabilize maturevascular networks (Saunders et al., J. Cell Biol., vol. 175, pp.179-191, 2006). Indeed, at the end of day 3, stabilization of thevascular networks formed within AHA hydrogel is evident as indicated bythe increased expression of TIMP-2 and TIMP-3 by ECFCs (FIG. 16D).

Functionality of ECFC Vascular Networks within AHA Hydrogels

To determine the functionality of the engineered vascular networks,3-days matured and stabilized vascular constructs were implanted intonude mice. Histological analysis revealed that the AHA hydrogels werecompletely degraded by 1-2 weeks in vivo, replaced by active or deadmacrophages and tissue ingrowth (FIG. 17A). Blood vessels in varioussizes are found at the periphery and center of the hydrogels (FIG. 17B).Most of the microvessels at the center of the hydrogels are positive forhuman CD31 and perfused with blood cells, indicative that the implantedhuman vascular networks were able to anastomose with the hostvasculatures to form functional vessels (vasculogenesis) (FIG. 17C).Some of the microvessels contain both human CD31+ cells and host cells,demonstrating that the implanted ECFCs participate in the angiogenesisof the host vasculatures as well (FIGS. 17C and 17D).

1. An vascular lineage cell growth medium comprising a hydrogel having astiffness that allows formation of capillary-like structures (CLSs) withextended vacuoles and open lumens from vascular lineage cells, thehydrogel comprising a crosslinked mixture of an oligosaccharide and acrosslinking moiety, wherein the hydrogel is cleavable by an enzyme; anda growth factor in an amount sufficient to promote vasculogenesis andtube formation, and sufficient to stimulate MMP production in vascularlineage cells.
 2. (canceled)
 3. The medium of claim 1, wherein thecrosslinking moiety is a peptide.
 4. (canceled)
 5. (canceled)
 6. Themedium of claim 3, wherein the peptide comprises the sequenceGCRDGPQGIWGQDRCG.
 7. The medium claim 1, wherein the hydrogel furthercomprises an adhesion promoter in an amount sufficient to promotevascular lineage cell adhesion to the hydrogel and promote vacuole andlumen formation in vascular lineage cells.
 8. (canceled)
 9. The mediumof claim 1, wherein the hydrogel has a Young's modulus between about 10Pa and about 500 Pa.
 10. (canceled)
 11. (canceled)
 12. The medium ofclaim 1, wherein the oligosaccharide is modified with an acryl group.13. (canceled)
 14. The medium of claim 1, wherein the oligosaccharide isselected from the group consisting of hyaluronic acid, dextran, alginateand chitosan.
 15. (canceled)
 16. The medium of claim 12, wherein theoligosaccharide is acrylated hyaluronic acid.
 17. The medium of claim 1,wherein the growth factor is VEGF, TNFα, SDF1-α, bFGF, angiopoetin-1,PDGF, TGF-β, PIGF or combinations thereof.
 18. (canceled)
 19. (canceled)20. The medium of claim 1 further comprising vascular lineage cells. 21.(canceled)
 22. (canceled)
 23. A method of inducing vascularizationcomprising culturing vascular lineage cells in a medium comprising ahydrogel and a growth factor, wherein the hydrogel has a Young's modulusbetween about 10 Pa to about 500 Pa and the hydrogel comprises acrosslinked mixture of an oligosaccharide and a crosslinking moiety. 24.(canceled)
 25. (canceled)
 26. (canceled)
 27. (canceled)
 28. The methodof claim 23 wherein the oligosaccharide is acrylated hyaluronic acid.29. The method of claim 23 wherein the hydrogel further comprises a celladhesion promoter.
 30. (canceled)
 31. A method of preparing a vascularnetwork comprising: chemically bonding an adhesion promoter to anoligosaccharide; combining the chemically bonded adhesionpromoter/oligosaccharide with at least one growth factor; adding acrosslinking moiety capable of forming a hydrogel with theoliosaccharide to the combination; and curing the combination to form ahydrogel; wherein vascular lineage cells are added before, during orafter curing; and then incubating the vascular lineage cells to form avascular network.
 32. The method of claim 31, wherein theoligosaccharide is selected from the group consisting of hyaluronicacid, dextran, alginate and chitosan.
 33. (canceled)
 34. The method ofclaim 32, wherein the oligosaccharide is acrylated hyaluronic acid. 35.(canceled)
 36. (canceled)
 37. The method of claim 35, wherein thecrosslinking moiety is a peptide comprising the sequenceGCRDGPQGIWGQDRCG.
 38. The method of claim 31, wherein the adhesionpromoter is a peptide that includes an RGD sequence and a reactivefunctional group that is capable of bonding the adhesion promoter to theoligosaccharide.
 39. The method of claim 31, wherein the growth factoris selected from the group consisting of VEGF, TNFα, SDF1-α, bFGF,angiopoetin-1, PDGF, TGF-β, PIGF or combinations thereof.
 40. (canceled)41. (canceled)
 42. The method of claim 31, wherein the hydrogel has aYoung's modulus between about 10 Pa and about 500 Pa.
 43. (canceled) 44.A method of growing blood vessels in vitro by incubating vascularlineage cells with a medium according to claim
 1. 45. A method ofpromoting vascular growth in a subject by contacting the subject with amedium according to claim 20.